Nursing Concept Map

[applause] bob wildin:thanks very much. can everybody hear me all right? okay, good. so, i’m bob wildin. i’vebeen here at nhgri about nine months. i’m an m.d. clinical geneticist with a backgroundin research and software development. and i came here to help nhgri do whatever is neededto bring genomic healthcare into general medical practice. and education is one of those things,and so this is really actually very important to me and to us at the genomic healthcarebranch. and i want to thank all of the people who’ve really helped put this together andinvited me to do this. and i want to thank you guys for being here because you’re reallyimportant in all of this as well. so without

further ado -- which button, here -- okay. so the general plan here is to first talkabout dna. that’s an acronym; we’re in the government so we have to talk in acronyms.but this is a -- and then genome, and then we’re going to talk about replication andvariation. we’re not going to talk about gene right away, because i think all of thisis actually really important to understand, all of this part down here. we have a twohour session here. i think we’re going to take a break in between the first hour andthe second hour. and i’m not sure exactly where the break is going to fall, but mayberight in here. and then we’re going to talk about some more complex and human-orientedissues in the second half. so that’s the

general plan. okay, so what about dna? and let me say first,please interrupt me, raise your hand if there are questions, if i’m going to fast, ifi’ve said something that isn’t clear. if i see you guys looking at each other, youknow, please stop me, this should be a little bit interactive. we’re a small enough group,we can do that. okay so dna is where? so it’s in every cell. and a cell is kind of likean encapsulated system. so, this is a diagram of a cell that’s been cut open. and insidethis little styrofoam-looking ball is the nucleus, and it’s been kind of cut open.and most of the dna in the cell is inside the nucleus and is packaged as chromosomes.so that’s the most commonly thought of place

of where the genetic material resides. butthere’s another hidden place, anybody want to venture where that is? female speaker:mitochondria. bob wildin:mitochondria. okay, so the mitochondria are over here. there’s more than one mitochondriaper cell. the mitochondria are inherited from mother, not from father, which makes the inheritanceinteresting. and so that’s pretty much what we’re talking about. so what is dna? anybodyrecognize this figure up here? female speaker:[unintelligible]. bob wildin:okay, so that’s the x-ray crystallography

picture of dna. and that was critical in identifyingthe structure of dna. so dna is a chemical, it’s deoxyribonucleic acid, and we’regoing to go into more detail on that. it has a structure which is this double helix. andit contains information. and this is my little panel to show that, you know, a string ofany kind of letters doesn’t necessarily have information, it’s the order of thoseletters that creates information. and so the sequence is the order of bases in the dnastrand. and the genetic code is the way the information is interpreted for protein sequence.but there’s lots more information in the genome, we’re just not sure how to interpretit. okay. because it’s not based on this genetic code.

all right, so the basic structure is two strandseach -- of the sugar phosphate backbone and we’re going to get a little more geeky aboutthis in a few minutes. and the base pairs are bases that are attached prevalently toeach strand, and then attached to each other via hydrogen bonds, and those are called basepairs. all right. so, i put here how to get it, and i think you’re going to do this,or watch the full video tomorrow morning, but i just wanted to give you a quickie here. [clip playing] he’s our boss.

okay so, you’re going to do more of thattomorrow. so let me go back to this. any questions about where dna is? even in the strawberries,okay. so, and how fragile is dna? anybody have any idea as how fragile it is? no? canyou, you know, pick it up off the microphone in front of you, it's been there for a while,yeah. so dna is not fragile. it’s actually very durable stuff. you can split -- the easiestway to disrupt it is split down the middle, split the base pairs down the middle and maketwo strands. you do that by heating it up. but if you let it cool down slowly, it willgo back together. okay? so that really doesn’t destroy it. you can tear it, shear it likethat, and cut across this way, and you can break it into smaller pieces, but you’renot destroying the dna. you’re only destroying

the information, you know, in the case ofsort of ripping a page in half of a book; you can still read most of it. all right?so, and it can stay around actually for thousands of years. so it’s actually really durablestuff. and that’s something that most people don’t realize. i’m going to talk about some confusing pointsabout the dna structure. things that, i think people in general get confused about, andparticularly students who are learning it for the first time. one is this sort of doublehelix. so the double helix is the structure. and that’s what was generated by this x-raycrystallograph. and it has these two strands that are a helix, a round helix, they’rejust kind of curving around each other. so

i think of the double helix as the structure.and then the strands have opposite direction, which we’ll go into more in the next slide.the double strands, this is double-stranded dna versus single-stranded. if you split itdown the middle by heating it like i talked about, then you get two single-stranded molecules. and there’s confusion between the doublestrands and having two gene copies versus one gene copy. so, this single molecule ofdouble-stranded dna will be one copy of a gene. and in diploid organisms like we are,in eukaryotes, we have two copies of almost every gene. okay? so you actually have twoof these, and that’s the two gene copies that we talk about when we talk about thecopy we inherit from mom and then the copy

we inherit from dad. okay? so those are conceptsthat double-stranded is the structure of the molecule itself. and then the two copies isbecause we have two copies of these. okay? all right, great. so now we’re going toget a little bit geeky and get into chemistry. okay? so double-stranded dna-based pairing.it’s deoxyribonucleic acid, so if you cover up the deoxy and say, well what’s -- andtry to break this down so it’s ribo, which is about ribose, right? it’s this sugarmolecule here in the sugar phosphate backbone. that’s the blue things that form the backboneof the helix. and nucleic means it’s found in the nucleus, right? they sort of namedit after where it was found. and then acid, it’s actually a mild acid. and you can tellthat if you’ve ever seen dna fragments separated

on an electrophoretic gel, right? it movestoward the positive, if i’m getting this right, yeah it moves towards the positiveterminal, right? because it’s negatively charged like an acid that’s been ionized.okay? so it’s really a chemical, it's a complexchemical but it’s a very predictive, a predictable chemical. so it has these sugar moleculesthat are connected with phosphate molecules. and there’s a little bit more detailed showingin a minute. and then attached to the sugar molecules, sort of pointing into the middleof this helix between the backbones are the bases. and there are a number of differentways in which we diagram the bases. and this is one sort of cartoonish way to diagram thebases that demonstrates, by the means of the

sort of lock and key fit, that adenine tendsto pair with thiamine, and guanine with cytosine, and so forth. okay? and that these two strandsare really held together only by the hydrogen bonds between the bases. okay. the other thing before i move on to that isto note that these two strands are actually moving in opposite directions. not moving,i should say they have an orientation. so these sugar molecules, if we look at thispoint, is pointing in this direction, and the sugar molecule is pointing in this direction,so they’re actually really identical chemicals. so if you take this side and turn it overthe other way, it will be pointing the other way, just like that. okay? it’s differentthan, say, having those hooks that you hook

your garden gate with that has a hole anda hook. those hook together, they're complementary, but they’re really not identical kinds ofthings. that’s one of the really cool things about dna, is they’re complementary, butat the same time identical, if you ignore the base pairing for the time being. so, the other cool thing about dna is it hasthese bases that base pair with each other. and the base pairing is by hydrogen bonds.and what i think is really cool about this is that hydrogen bonds -- hydrogen if youget into real chemistry, is a univalent molecule, which most people think, okay hydrogen isunivalent -- that means it can only form one bond, right? but hydrogen does this reallyneat thing called -- it has a covalent bond

it forms there, but it also can form theseweak bonds, these hydrogen bonds, with a particular oxygen or nitrogen. and the cytosine and guaninepair have three opportunities for hydrogen bonding, whereas the thiamine and adeninehave two opportunities for hydrogen bonding. and what that means is that this pair actuallybinds tighter than this pair. okay, so if you have a stretch of a’s and t’s in arow, that’s going to be easier to pull the two strands apart than if you have a stretchof c’s and g’s. okay? just something to keep in the back of your mind for maybe someof the stuff that’s coming up later in the course. all right, it seems to me there wassomething else i was going to talk about. any questions about this? okay, all right.

what is a genome? so, is this a genome? no,this is a wallaby. i put this up here because we don’t really have a good way to diagramwhat a genome is, right? there’s no sort of picture of a genome and in fact in tryingto find one, this is the closest i found. all right? so this is an electron micrographof a genome of a bacteriophage. anybody knows what a bacteriophage is? so it’s a virus,this tiny, tiny, tiny, tiny, virus that infects bacteria. that’s how tiny it is. and ithas one dna strand. and it has just enough information to tell the bacteria, “makemore of me.” okay? so that’s a very, very simple type of genome. so, the human genome,on the other hand, is really in a lot of ways the same. it’s a huge amount of dna, it’sa lot more dna, but it's saying, you know,

how to make more of me. okay? and it’s madeof dna, which we’ve gone through. it’s all of the genetic material in the nucleusplus the mitochondrial genome. it has molecules of dna that contain the coded instructionsfor how to build, maintain and replicate a human being. okay? now we have an interviewhere i want to show you about how kids view genes. okay, so if you’re in high school, you knowit may not be cake, it may be sex or something else so -- [laughter] it’s really cool. okay. so, let me get outof this. and -- that video was taken at the

“unlocking life’s code” genome exhibit.and i don’t know whether you’ve already been told about that. it was an exhibit atthe smithsonian, and now it’s traveling around the country. and it was developed byour folks here, and along with the smithsonian. and it’s in st. louis now, and then nextit’s going to portland, oregon. so, if you’re in those areas, please keep an eye out forit. but i thought that was a really neat way of a kid really getting what a genome is.okay? human genomes are not identical in anyonebut twins. okay? they’re very close to each other, but they’re not identical. dna -- thegenome always contains both benign variation, a variation that doesn’t cause any problemfrom the medical or life standpoint; and variation

that can cause or contribute to diseases.okay? so all of us carry genetic changes that, in the right circumstance, might cause disease.it may mean that we have to have two copies of that particular variant in order to causedisease, and we only have one copy, so we’re a carrier. we’ll talk about that more later.but we all carry those kinds of things. okay? so nobody is completely clean, so to speak.and the human genome is really big. its 3.3 billion base pairs, which is a lot of zeros.and it’s actually twice that because we have two copies. right? we have a copy weinherited from mom, and a copy we inherited from dad. okay? lots and lots of sequence. all right. the human genome, and eukaryoticgenomes, well all genomes in fact are organized

into what are called chromosomes. so a chromosomeis basically one strand of dna. it can be really, really, really long, or it can beshort. okay? and the bacterial chromosome is a circle. okay? viral chromosomes tendto be single, short, linear molecules. and human chromosomes, like other eukaryotic chromosomes,are these really long pieces of dna that are packed together. so we’re trying to packa lot of information into a small package. we start from this, sort of the dna strand,and we add histones and then those stick together to form nucleosomes, and then we wind thosenucleosomes up, and we wind the wound up nucleosomes into tighter and tighter packages until weget something, at least during cell division in prophase, that looks like a chromosomethat we can see under the microscope. okay?

and just to take this opportunity to showthe parts of a chromosome. so this is a replicated chromosome, and it has two sister chromatids.and the ends or telomere, for you linguists, sort of telo like telephone, you know faraway, and centromere, central. and then the p arm and the q arm which is the short armand the long arm. the only way that i can remember the difference, it’s hard to remember,is to think of p as petite, okay, for which is french for short. all right, that’s howi remember it. and then the chromosomes themselves are packed into the nucleus there. so whenwe do a chromosome study in medicine or in the laboratory, we get a picture that lookslike this. we put the cells through cell division, and we catch them at just the right time whenthe chromosomes are really condensed like

this, and then we take a picture of it underthe microscope. and then we used to actually literally, you know, print the picture onpaper, and then we would take scissors and cut out the individual chromosomes, and stickthem onto a piece of paper that had these numbers on them so we would array them. so there are 23 pairs of chromosomes. andthey’re basically organized by the length, okay? so, before we had this ability to barcodethem, sort of stripe them, they just looked like pieces and we would order them by theirlength. okay, so that’s where the chromosome numbering system comes from. and now we haveability to -- well for many years now -- to add a stain which shows different stripy areasand that allows us to sort of see which chromosomes

that are almost the same size, how they'redifferent, and which ones are actually which. okay? so, 22 pairs of autosomes in the yellowbox. and one pair of sex chromosomes, in boys that’s and x and a y, in girls it’s twox’s. okay? this is probably not new to you at all. we’re just reviewing a lot of stuff.they’re packages of dna, and they have a consistent structure within a particular species.okay? so i threw -- this next slide is not in yourprint, but this is a comparison between human, chimp, gorilla, and orangutan species. andit’s the same chromosome sets, but in diagram form. okay? and what you see is that a lotof places the banding system is entirely the same. and in other places, there are littledifferences here. and then sometimes the chromosomes

are broken into two parts. so here’s, youknow, here and here are two different chromosomes that represent the human chromosome numbertwo in the other apes. okay? so, a lot of it is the same material, but it’s kind oforganized a little bit differently. all right. human genome project, whose heard of that? yay, okay. does it come up in your classesa lot; does it come up a lot? okay. so, i’m not going to talk a lot about it, but basicallyto say that the human genome project was this really big deal that came out of nhgri. andthe concept was that we’re going to sequence the entire genome. we’re going to get thedna information from the entire genome, starting with the genes really. and we’re going to-- that’s going to push us forward in terms

of knowledge and ability. and i will tellyou that, as a scientist who was trying to get grants for simple, direct, basic diseaseresearch at the time, i was not very happy about this, because it costs a lot of moneyto do this. and it was brand-new technology, and it was risky. and what they were doingwas saying, you know, "we believe that, after we get all of this information, it’s actuallygoing to be useful." but they haven’t proved it yet, right? and if i were applying fora grant at the time and said, "oh, i’m going to do this big thing, and afterwords i’mgoing to have all this information, and it’s probably going to be useful," it would’vegotten rejected. but these guys were actually really smart. and i’m really glad they didit, even though i was not very happy at the

time. so, because it really has panned outto be a tremendous boost to, not only genome research, but also research for all kindsof biology and human diseases. so it’s become really this amazing tool rather than a resultin itself. it’s became a terrific tool. so here’s kind of the timeline. and thenthis human referenced sequence was complete. and we really were excited about that. andover time, we realize that yes that was really great. but yes, there are actually holes,and yes this piece doesn’t actually connect to this piece. so this project actually issort of continuing in some ways. in that it is refining the information and the qualityof information that was gathered at that time. okay. all right, this is sort of my timelinedescription of what was actually done. so

the first thing was done, what was not tosequence because we didn’t have the technology to sequence the whole genome. the first thingwe done was to map the genome. the reason i erased this is because people can confusethese two things. and i think that’s helpful to distinguish sequencing the genome frommapping the genome. and mapping the genome was in the early part of the project, where,through sort of traditional genetic techniques, applied in very high throughput technologysystems, we were able to put sort of markers all along the genome. okay? sort of mile markers. and so we were able to identify kind of wherethe towns were, and where the major population centers were, but we didn’t have the informationabout every spot on the road in between. okay?

and we are able to tell which towns linkedup with which towns. and then we begin to get more detailed by sequencing, and beginto get this idea that, yes there were houses there, there were trees, there were otherthings in between by sequencing that we were able to get to. and then, we were able toget down to the point of seeing even sort of the shingles on the house. okay? so let’scall the shingles on the house the base pairs, and the house would be a gene. so that’skind of a metaphor for how the genome project progressed. so the output of the genome project was hugeamounts of sequence data. all right? sequence data we store in text files basically, wherewe put down the base sequence acgt. all right?

this is a very simple file format called fasta,f-a-s-t-a. and this is the sequence of one gene called foxp3 which is when i used towork on years ago. and what this slide doesn’t show you is that there are about six morescreens full below this one just to describe this one actually quite small gene. all right?so when you’re talking about 20-plus thousand genes and most of them are going to be biggerthan this, and when you’re talking about the gene part of the genome being just onepercent of the genome, you’re talking about a lot of sequence. okay? so we’re talkingabout information technology being absolutely critical for this process. all right, dna replication. all right, sodna replication is important because, for

a lot of different reasons. but let’s basicallygo through it. so we have a double-stranded dna, and we want to make two copies, we wantto make one copy of the double-stranded dna into two copies of double-stranded dna. andthat’s important because we need to do that before we can duplicate cells. before we canmake two cells out of one cell. before we can make two organisms out of one organism,per example. so, the process is first that you have to unwind the dna, and then you haveto take an enzyme, called dna polymerase, so dna is a polymer, right? it’s a bunchof sugar phosphate backbone’s and bases, it’s a polymer. and ase, that suffix ase,generally means it’s an enzyme. so dna polymerase is an enzyme which makes a polymer calleddna. okay? and it does that by attaching to

a single strand of dna with a little shortsequence that’s attached to it. and it sees that open thing and all of these open basesas an opportunity to add more bases. so if you have the dna polymerase, you have thesingle strand of dna and you have a starting point, then all you have to add his nucleotides.and the dna polymerase will say, “okay ready, i’m ready to copy,” and it copies. allright? so you need dna polymerase, you need nucleotides, you need template, you need atp.what’s the atp for, anybody know? male speaker:energy. bob wildin:energy. it provides the phosphate and the sugar phosphate backbone. okay? plus the energyto make those bonds form. all right? it’s

a directional process. it goes from five primeto three prime. all right? and the information is preserved, because a base will only beadded if it’s complementary to the base on the other strand that’s being copied.okay? so it’s complementary, but it’s the same information, right? you can copyit again on the others, you know, copy your new strand back to the other strand, and youhave exactly the same thing. all right, questions about that? yeah? female speaker:this may be very low-level, but i get confused about when is this happening, like in a cell?like is it at development, or throughout life when, like, at what -- maybe that’s -- it’svery simplistic --

bob wildin:no, i think that’s a great question. so the question is, when does dna replicationhappen? and it basically happens any time you need to divide the cell. right? so ifa cell is just sitting there like most of our brain cells, they’re not dividing, they’resitting there doing stuff but they’re not dividing, okay? so whenever you need to makeanother cell, two daughter cells out of one cell, you need to replicate the dna becausethe cells need those instructions. okay? we’re going to go through later, mitosis, whichis that cell division process. and then even later we’re going to go through meiosis,which is the process that makes germ cells like sperm, an egg. okay? so this is a videoof -- i think i have to click to make it go

-- it’s an animation, but i think it -- so i think that’s kind of mesmerizing inaddition to being informative. so, what they were talking about was that when you’reunwinding the dna in one direction, remember the strands are in two different -- are goingopposite to each other in terms of their orientation, so one of them can be copied five prime tothree prime directly, and the other one, you have to go a little ways down and copied backwards.okay, because the polymerase only goes in one direction. so you have the replicationfork, and the leading and lagging strands. so those are terms that are used that i thinkyou’ll hear about. and i thought the video

was kind of helpful in showing that.all right, how do we use dna replication? i think this is really, really important aswell. so we use it for technology, right? i mean, it’s been completely co-opted, notjust to divide cells, but to do all kinds of stuff in the lab in the test tube. all right, one of them is -- most dna sequencingtechnologies are based on dna replication. all right, and this background here is notthe latest dna sequencing technology, but one that’s probably 15 years old, whichin all of these different colored squiggle lines are the lines that represent the positionsof a’s, c’s, g’s, and t’s. each one with a different color. okay? and basically,you're sequencing down the way and stopping

every time you reach an a, every time youreach an a, every time you reach an a, and that’s what these peaks represent. okay?and you have another tube where it stops only when you reach a c, a c, a c, a c, and that’swhat the c peaks represent the blue ones. okay? so there are lots of different waysthat you can use the sequencing technology. who’s heard of pcr, preliminary chain reaction?wow that’s impressive, okay. pcr is a technique which was developed in the mid-1980s. basicallyto take an input dna, and if you know the sequence, you can design starting points forit, and then you exponentially replicate the dna in a test tube. and it’s a really coolprocess. and it was another one of those sort of real kick starters in terms of the technologythat allowed us to do a lot of the stuff,

including the sequencing that was done inthe human genome project. mutation detection techniques, some techniquesthat use the polymerase to go down the line until they stop because they’re missinga particular base. dna diagnostics, a lot of them are based on replication. and thenthings like, you know, reconstructing ancient genomes, where you only get little tiny fragments,but the fragments may overlap, and you can use polymerase to pull them together. i’mgoing to go back here and just say, so this dna polymerase plus nucleotides plus template,you can do in a test tube. all right, you can do it in a tiny little test tube, it’sactually really easy. and one of the cool things is that you use a dna polymerase frombacteria. okay? bacteria make it, you extract

it from bacteria, you put it in a test tubewith human dna, does it matter? no, it doesn’t matter. dna is dna, it’s a chemical. allright? the information in the dna may encode human genes as opposed to bacterial genes,but the polymerase doesn’t know that. it’s got a job to just replicate it. okay, that’sit. i think that’s pretty cool. all right, replication versus mitosis. sothis is another area where people can get bogged down, because we’re talking aboutmaking two of one thing in both cases. so replication is the process of making a copyof dna, and we’ve just been talking a lot about that. mitosis is the process of replicatingthe genome and separating the two copies in cell division. okay, so here’s a diagramof mitosis. and you’re starting out with

one diploid cell, so one cell with two copiesof both genomes, mom’s genome and dad’s genome. and the dna is all spread out, it’snot condensed, and that’s called chromatin. you have 46 chromosomes, two sets of 23, or2n. and then what you get out at the other end is two diploid cells with 46 chromosomes,each with 2n. okay? so you take one cell and you make two cells. that’s mitosis. allright? the steps are to condense the dna into chromosomes, replicate the dna, you have asister chromatid, and now you have two sister chromatids on each set of chromosomes. youorganize them, you line them up in the middle, and you pull them apart into two sets, andsomehow they know that only one of each chromosome goes into each daughter cell. and then intelophase, you’re separating the cells,

and you get your final cells there. so one other point that often is confusingfor students, i think. okay, so this is my diagnosis [spelled phonetically] -- mitosisis what you want your money to do, right? okay? just, you take one and you get two.all right? so centrosome and centromere, so this is a sort of anatomy in the cell kindsof things. centromere is that place in the middle of the chromosome that’s constrictedokay, and where the two sister chromatids stick together until anaphase when they’repulled apart. the centrosome is this little microtubule organizing guy that’s out inthe cell, and it replicates as well, so you get two during mitosis. and it basically formsthe organizing point for the microtubules

that go out and grab the centromeres fromeach sister chromatid and pull them to opposite ends. so centrosome and centromere. okay. in a replication is not perfect. okay? dnareplication machinery has a proof reading function. and sometimes it’s built intothe polymerase itself, and sometimes it’s an associated protein. and the idea is thatit doesn’t want to make mistakes, because if you, you know, garble everything, thenyou don’t have instructions anymore, you have garbage. right? so you want to be prettydarn good at replicating the dna, because it’s important information. okay, it’slike, you know, sending your email and having it come with every third, you know, lettermessed up. it loses information and that’s

not good. but nobody’s perfect, okay? it’snot perfect, the replication isn't perfect. and sometimes, that may not be a bad thing.so, i want to stop for a second, and have you pair up with whoever’s near you, andtalk about why, just for two minutes, talk about why it might be important for replicationnot to be perfect. [inaudible] bob wildin:so you doing okay? all right. okay, wrapping it up. did we figure it out? at the far endof the table, do you want to start? no? anybody? female speaker:we thought [unintelligible] would be perfect because it can adapt to changes [spelled phonetically].

bob wildin:okay, yeah. does anybody want to dot the i’s and cross the t’s on that idea? female speaker:genetic variations that could lead to evolution as a helpful mutation. bob wildin:okay great, so if you think about it, if you think about that original dna molecule back,you know, at the origins of life, what would happen if it were perfectly replicated allthe time? you would never have variation on which to select, or the environment, for advantagesin the environment, to improve. so basically, the fact that proofreading isn’t perfect,is what makes evolution run. okay? and sometimes,

this is hard to explain to people, but thisis the way i explain it. so, you know, why do people get mutations that cause breastcancer? well, replication errors are part of life, and they’re part of life in partbecause, without them, you don’t get this process of evolution. okay? any questionsabout that? okay, great. you guys are really smart. okay, so there are other things other thanproofreading errors that cause dna to not be completely --have total fidelity in, youknow, living and life. so there are mutagens, like chemicals. and i put this thing up, ifound this on the internet. i think it’s kind of interesting. so in california, youknow, you always see the labels that say,

you know, “contains carcinogens.” right?and then you eat off of it. bob wildin:so ultraviolet light is something that dna is very sensitive to. okay? the bases in thedna structure, getting back to physical chemistry, actually absorb uv light. and in the laboratory,that’s how we measure how much dna is in solution in our test tube. we put it througha spectrophotometer that shines uv light on it and says, “well this much uv light wasabsorbed.” and we then calculate from that how much dna is in that tube. all right? ifyou have a lot of uv light, uv light will damage the dna, because that energy is absorbedand eventually, it breaks the bonds. okay? now there are in organisms' specific machinerythat goes around and looks for the type of

damage that uv light produces, and repairsit. it’s not perfect either, okay? that’s why i have age spots. all right? so we haveuv light damage for dna. ionizing radiation, that’s like x-rays, all right if you gettoo many x-rays you can damage your dna. and then the things that are more complexlike dna repeat instability, so there are segments of complex genomes like human genomesthat are actually repeated. so there are, you know, 2000 bases that are kind of in order,and then they’re repeated again next to that, and they’re repeated again next tothat, and they are repeated again, they can be repeated hundreds of times. okay? but thoserepeats can become unstable, or they can form loops during the replication process, becauseif they’re repeated during the replication

process, those two strands can get intertwined,and you’re not sure whether you’re the part down here or the part down there, andyou can actually delete or duplicate big segments of dna. there’s a category of human disease calledtriplet repeat diseases. and those are just cgg, cgg, cgg, for example. and those tripletrepeats, in certain contexts, can become expanded. so instead of having 36 repeats or a normalrange between, you know, 18 and 50 repeats, you have 300 repeats okay? one example isfragile x syndrome, which you may have heard of, huntington’s disease. okay? so you canhave diseases, a variation that results in repeat instability and expansion or contractionof repeats, and that actually changes the

gene expression, or the gene sequence, andcauses disease. all right, so this slide is supposed to tell me that we’re going totalk about variation, okay? so there’s all these chairs, and they’re all orange, sothey’re all the same. right? no, not quite, they’re a little different. they’re allchairs and they’re all orange, but they have variation. so variation can be brokendown into kind of two grand categories. so this is a variation that darwin observed,it’s a variation phenotype. okay? so what things look like on the outside, what arethe characteristics that are observable? and then there’s genotype variation, or whatare the differences at the actual gene level? okay? and we're going to go over that in alittle more detail.

the variation has its origins in the genotype,most of the time. and there are consequences to variation. sometimes there’s no consequence.you can change a base, you can change the encoding amino acid, and you have no consequencewhatsoever because it really doesn’t make any difference in the environment of thatorganism. you can make things worse for them, you can make it better. okay? worse is notgood, better is good in terms of evolution. and those differences may be environment dependent-- which i may not have spelled right. we talked about evolution and then the phenotypicvariation. and disease is one category of phenotypic variation. okay? variation meansit is found to vary, that’s all it means. it’s defined in the genome by referenceto a standard, which may or may not be normal.

okay? so, back on the human genome project, thestandard, the reference sequence when it was generated was actually a composite of severalindividuals. but, it was their sequence. and it, as i said earlier, it probably containeddisease gene variants that were not considered normal. okay? there are lots and lots of normalvariation, much more normal variation, but there are variations that may not be normalin the reference sequence or the standard sequence. variation occurs normally withoutregard to functional consequence. that’s what we’re talking about, so the variationhappens due to replication errors. the errors in general don’t -- they’re not made becausethey need to be made, they’re made because

they happen. and then they are selected throughevolution. and it’s subject to selective pressures. and interestingly, a variationcan occur, originate in one member of a population, and then the descendents of that individual,at least some of them will inherit that and pass it on. okay? so, that results in frequencydifferences between different population lineages. can anybody explain that better? okay. orgive an example? so somebody’s going to try it, go for it. female speaker:it’s kind of a founder effect, is that what you’re kind of describing? so like if you’re-- if you carry a disease and you’re on an island in the caribbean, and you pass itto all of your children or whatever. you have

10 kids, then that would then spread the diseaseif it’s like a single -- is that what you’re kind of getting at? bob wildin:yeah, another example might be populations that tend to stay together. certain tribesor populations, the one that we think we hear about more often in medicine is ashkenazijewish populations, who -- female speaker:the amish are a good group as well. bob wildin:the amish is another one. okay, so if that particular variant, which originated in thatpopulation, if that population is not strongly outbred, then it will remain associated withthat population, have a higher frequency in

the population than it does in another populationthat didn’t have the opportunity to interbreed, okay. female speaker:i have a question. bob wildin:yeah. female speaker:[unintelligible] variation through the next generation, does it have to be the reproductivecell or can any of the somatic cells pass on the variations, too? bob wildin:only the reproductive cells. so if it’s not put into the sperm or the egg, it doesn’tget passed on. right? so, and what you may

be getting to is sort of cancer mutations.so, if the cancer mutation -- we differentiate -- actually, it's a really good question -- sowe differentiate cancer mutations between those that are somatic, meaning they’rein the body cells, that can cause cancer. or those that are germ line, meaning theyare in all our cells, including the ones that contribute to egg and sperm, that get passedon. okay? and it’s the latter that are the form for hereditary cancer predispositionsyndromes. okay? so like brca1 and brca2, as an example, where you have a gene thatpredisposes you to breast cancer in all your cells, you get breast cancer, ovarian cancerat lower frequency than other cancers because those are more sensitive to particular mutation.but in your germ cells, they’re going to

get passed on at 50-50 chance to your offspring.does that answer your question? okay. a really good one. yeah. female speaker:what i'm trying to [unintelligible] i'm not sure what the process is to say not to worryabout these, or to worry about these, should we test other people in the family, and notto test other people in the family as it comes up with [spelled phonetically] -- bob wildin:that’s a really good question. and we actually struggle with this a lot. and we’re doinga lot of work to try to sort out you know exactly what that process should be. and oneof the things that we do is to kind of divide

those variants that we get back on a genomereport, a panel report, into categories that say this is how much we know about this particularone. so, like a lot of the brca1 and 2 mutations we’ve seen before. it’s very clear thatthere associated with an 80 percent risk of breast cancer for example. now we can sayclerically, that’s a pathogenic mutation. that’s a mutation or variant that causesdisease, all right? there are others that seem to, if you look at the biology of it,they say, "well, they stop the protein synthesis," which we’ll talk about later, but they don’t-- we really don’t know for sure that they're associated with this condition. okay? andthose are called variants of uncertain significance. right? and we don’t know what to do withthose. we’re hoping that as we gather more

information, we will know which one of thoseare normal variants because they are at a significantly -- they're seen in a populationthat doesn’t have a high risk for breast cancer, for example. okay? female speaker:what information on genotype do you try to gather that's [spelled phonetically] -- bob wildin:when we do the testing? is that -- female speaker:no, to say whether this is going to be a problem or not. bob wildin:so we -- there’s a wide range of information,

so we’re looking at whether the particularvariant is predicted to cause a difference in the protein that is bad for the protein.right? or it may be bad enough that the protein isn’t even made or it's only half of itis made, in which case it can’t form into a full functional protein. okay? we’re lookingfor association with families who have passed this down through generations. and in everygeneration, there’s been a high risk. those who have that variant have a high risk forcancer, and those who don’t have that variant don’t. okay, so that’s a different kindof information. all right? we’re looking in model organisms, which i think you’regoing to hear more about too, whether, if we introduce that mutation into the modelorganism, is there an increased chance of

cancer or whatever the phenotype is in thatmodel organism? we’re looking in chemical assays in thelab to say if you introduce that mutation into cells in the laboratory, do they behavelike cancer cells, for example? or do they do things that only cancer cells do, or makechemicals like cancer cells do? so there’s a whole wide range of information that’strying to be put together to make that determination. and then on the other hand, part of the precisionmedicine initiative is to sequence lots and lots of people. the whole genomes of lotsand lots of people, of all ages and figure out which variants are out there that aren’tdisease causing. so we don’t know completely, what’s the limit of normal? okay? what variationsare normal? so if we can definitively say

that variation is not associated with canceror anything else, then we can check it off. and we don’t even have to report it to youor to the patient. does that answer your question? yeah. okay, so here are some other confusing points.and we’re getting close to the hour so -- i can’t remember exactly where i am -- sothese are the things that are not synonyms with variation. okay? mutation we used touse, and i still do, because i’m an old fart. mutation we used to use to describesomething that was different, right? and especially in disease. so a mutation is -- but now weuse the term variation. okay? for this very reason, because a lot of times we’re notsure whether it’s a disease-causing variant

for not, all right? we call it a variation.so we reserve the term mutation now for the molecular and chemical processes that resultin new variation. otherwise we try to avoid that. the other reason is because the termmutant, at least in high school, is not very well-received. okay? so it kind of has a negativeconnotation and we try to stay away from it. right? female speaker:unless you like the x-men. bob wildin:unless you like the x-men, that’s right, yeah. so polymorphism is a term, anybody runinto that term before? polymorphism, single nucleotide polymorphism is an example. andit’s a variation that exists in the normal

population at one percent frequency or higher.okay? that’s all it means [spelled phonetically]. and it’s an old term, you know, based onpopulation genetics, and it’s called a polymorphism. it’s assumed, but not proven, to have nodisease significance, at least for rare diseases. when we get into more common diseases, allbets are off. and an example is the single nucleotide polymorphism, so one base has changedto another base. okay, also known as a snip. a new variation from mutation is altered betweenthe last generation and this one. so somewhere in the production of that sperm or the egg,a replication error or something happened, and it’s a new change that isn’t in eitherparent, but it is in the offspring. okay? and that’s called de novo, which is oneof those dead languages from you.

marker -- remember i showed you the map duringthe human genome project, so markers are variations that were used to trace inheritance of dnasegments, or suggest linkage to diseased genes. okay? and the genome-wide association studiesthat you may hear about are an example of that, as well as linkage analysis, which isthe old way that we used to trace variations through individuals, through families. okay, so mutation. how does a mutation happen?and what are the different kinds of mutation or variation that can happen? this is a mutagenicevent. you have your double-stranded dna with complementary sequence on both strands here,and this is -- the event affects this g. and a number of things can happen, it can be deletedokay? and the other strand has to compensate,

the c goes away. you can have an insertion,you can add a base, one base or more than one base, as many bases as you want, okay,as an insertion. or you can substitute a base from g to an a, for example. and then, whathappens when you replicate that, the c becomes a t, right? okay. so that’s sort of on themicroscale, and on the macroscale, you can have big changes. these are chromosome diagrams, chromosomesticks, and you have a segment here that just gets deleted, and all the genes that are thereget deleted. and the result is then instead of having two copies of this chromosome, youhave one normal copy and one copy where some of the genes are deleted, and you only haveone copy of those genes. okay? that’s important

if the dosage of that gene matters for theinstructions for running the human body. you can duplicate it, which is the converse ofthat. you can take that segment and flip it over, put it back in the same spot, and that’scalled an inversion. that usually doesn’t cause a problem unless something at the breakpoints,at either end, is messed up. so that breakpoint was in the middle of a gene, or puts a regulatoryelement that drives a gene next to something that it doesn’t normally drive. okay? you can substitute, so you put something fromone chromosome onto another chromosome, you insert it. okay? the important thing hereis that everything is still here, inversion and the substitution, everything is in balance.right? the amount of genetic material and

the copy number of genes is all the same.right? so that can happen without a lot of likelihood of there being a problem. translocationis the same thing. you take something here and put it on one and then the correspondingreciprocal change, this piece goes down here and you get that reciprocal. okay? so in theindividual holding that, if it’s absolutely clean, without, you know, deletion or duplicationat the breakpoints, that may be okay. they may not have any disease, but when they passthat on, this variant is going to match up with its partner here in the germ cells andthe offspring have a chance of inheriting an unbalanced set of chromosomes. okay? all right, when should we take a break?

female speaker:we have two more in this section and [unintelligible]. bob wildin:okay, two more in this section, so we’ll finish up the section. all right, so genotypecodes for phenotypes. so i’ve been throwing these terms around, and so i’m trying todefine them now, a little bit late. so, genotype is the genetic code describing an individual.it’s that set of variations that we’ve been talking about that is unique to the individual.and the phenotype is the physical manifestations of genotype in the individual. okay? thisis sometimes a really difficult concept. for me, it’s like falling off a log, i can dothis in my sleep, but i’m a geneticist. okay? so, maybe you can tell me if there arequestions about these differences, genotype

and phenotype. no, okay. in this example down here, we have the phenotypeof fruit flies, normal wings, normal wings, normal wings, and wrinkled wings. okay? howdid he get wrinkled wings? in this case, it’s because of a homozygosity for a recessivevariant that’s present on both copies, both the parental copies of the chromosomes. okay?so the genotype in these two in the middle who are heterozygous is big w, little w, sothere’s one of the mutant alleles, and one normal allele. either way, that’s theirgenotype. they’re heterozygous for the small w, but there phenotype is exactly the sameas the homozygous with the large w. so, it’s a difficult concept. think about it some,maybe you’ve got it down, that’s great.

i wanted to make another point about variationand environment which i think we’ve talked about but here’s -- i just pulled this fridayoff of pubmed, and this is a krill, it’s a tiny little organism that lives in the sea,and is basically the bottom of the food chain for sea organisms. and what happens with climatechange, is that the sea water temperature changes. but this krill has been around foreons and is used to certain temperatures, and is optimized for certain temperatures.so, when you change the temperature of their environment, that creates a situation whereit may not be optimal for that organism. so it may screw up the entire food chain, okay?so i’m throwing out some kind of examples that your students might want to think aboutin terms of how this all impacts everything

around us. all right, more stuff about variation. it’sessential, it’s risky, it’s relevant, and it’s relentless. it happens all thetime. and it’s kind of like puberty, right? okay, so i’m going to stop there to takea break. how many minutes? female speaker:five minutes. bob wildin:five minutes? female speaker:okay, everybody, you have five minutes. you can use the restroom, get a coffee, whateveryou need to do. thank you. [end of transcript]