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OK, so at the end of lecture on Monday, just based on some
of the questions that people were asking me, there were a
couple of things that I definitely
did not explain fully.
It became pretty clear to me on that.
So I thought I'd spend a little bit of time recapping a
couple of the things we talked about on Monday just to kind
of clarify-- hopefully clarify--
some of those issues having to deal with when Kornberg did
this assay of where he figured out what DNA polymerase was,
what exactly was that assay, and where did the mutants come
from, and so on and so forth.
So I'm going to walk you through that.
And then, remember, Haroon actually asked this great
question about when I talked about DNA repair, about how
that works.
And so he's actually going to tell you what he's found about
how that works.
So we'll do a little bit of recapping, and then we'll get
into more of the stuff from lecture today.
OK, so I wanted to make something a little bit clearer
about this assay that was done to figure out how
to duplicate DNA.
And I said the very first time it was done, what was put in
was a template-- a single strand of DNA, and the
nucleotides, and all those other little co-factors, the
building blocks.
And the search was for an enzyme that would be able to
take that template, and use that, and build another strand
using the nucleotides linking this together.
And that was successful, and Arthur Kornberg--
so this is what went into that assay.
And then, first he put in an extract of bacteria just to
make sure the assay worked.
So if you put in all of the proteins that are in a
bacteria, and added them to this test tube that had the
single-stranded template plus those nucleotides, could he
get DNA out?
Yes, he could get long strands of DNA out.
Then when he fractionated that, could he get down to one
single protein that would work?
And yes, he could.
So that was one part of the assay.
But then I started to tell you about these mutants and then
about mutant extracts, and I think that may have been a
little bit unclear.
And so I wanted to just say a little bit more about that.
So in the first case, what went in here was a
single-stranded DNA template.
So just one strand of DNA, no proteins around it, nothing
complexed up into a double helix.
Just one strand.
And with that--
that simple thing--
that's how you could find one enzyme, one DNA polymerase,
that was able to copy that.
However, if you put in what the real template is-- and
that's double-stranded DNA that's wrapped up in the way
that would be in a chromosome--
and you try to do the same assay, and you try to isolate
DNA polymerase 1, it doesn't do anything.
It can't replicate that.
And it became clear that in order to do something, like
replicating a chromosome, it wasn't going to be one enzyme
could do the job.
You'd actually need to have a lot of different enzymes
contributing to that.
And in class, we talked about in the end, those enzymes that
were found.
So they're enzymes that open up that DNA, that are
helicases, that are primases, that then are the polymerases,
that are the topoisomerases.
So there are a lot of complex things in there.
And so if you're biochemist, and you're faced with this
process that you're interested in, think about that.
So say you had a chromosome, and you wanted to put in a
test tube all of these different fractions that--
and so you have this thing that you actually know is
going to involve 10 different proteins working together.
You have to be able to find those 10 fractions.
So you have to fractionate all--
you're blind.
You have to fractionate all the proteins in there, and
somehow pick the right 10 that, in combination, are
going to be able to do this.
You're never going to find them.
So that's going to be impossible.
You can find the one thing that does it, but trying to
find the 10 things, each of which you need for the
complex, it's not going to work.
So solution to this is to use these genetic mutants where
one thing is missing from that process, and then just try to
find what that one thing is.
And so I'm going to walk through visually a little bit
how to think about that.
So the first thing is actually, where do these
mutants come from?
I said there's these mutants, right?
And these mutants can't replicate their DNA.
So I should probably tell you a little bit more about this,
and introduce this idea of a conditional mutation.
So the first thing is, OK, so if you're bacteria--
let me move that away--
so you're a bacterium, and you have a chromosome.
And so, the first thing you do before you want to divide is
you replicate your chromosome--
they're circular chromosomes in bacteria.
And then you can divide, and each cell gets it own
chromosome, OK?
So that has to happen.
So what you're actually looking for are bacteria that
aren't able to divide, and make more bacteria, because if
they don't have a chromosome, then the bacteria
cell doesn't survive.
But here's the trick.
If you're looking for a mutant in a process that is
absolutely essential for your life, as soon as you find that
mutant, it's dead.
And so then, it's really hard to work on that mutant.
So there's another little trick here.
And if you think about bacteria--
so if you have bacteria, you can have them on a Petri
plate, and they can be in little colonies on a
Petri plate, OK?
So you can take a little piece of filter paper, or something
with a little handle on it, and basically make a little
replica onto a new plate.
You can also do this in tubes, but conceptually, it's the
same thing.
So you put this down on here, you pick up those little
colonies, you transfer them over here, and you have an
exact replica of these.
So you have identical bacteria in two different plates, OK?
Before you've done this, you've hit these bacteria with
mutagens, with x-rays or something that's going to
break their chromosomes, and induce mutations.
So now you grow some of these at 37 degrees, and some of
these at 42 degrees.
OK, so they're the same bacteria.
So they're bacteria, they're just--
bacteria don't have sex, so they're all
identical to one another.
So every bacteria in here is identical to every bacterium
in here, and everyone here is identical to everyone here,
although the ones in here are different
than the ones in here.
OK, so now you look at this, and you look for the ones
where they die.
And you say, OK, so this one may not live, this one may not
be able to replicate its DNA.
You have to do some more tests in-between there.
But at least you can work with this one, which is going to be
the same strain as this one.
OK, so conditional mutations are really, really useful in
genetics, because lots of the processes that we want to
study are essential for life.
We have to have some trick that lets us be able to
recover those things that would normally kill us.
And this is one trick where we just have a second copy of
that same bacterial colony, and that's the one that isn't
able to replicate.
OK, so this is what a conditional mutation is, and
the most common thing is to raise the temperature, and
look for things that can't do something at a high
temperature, but at a normal temperature, they're OK.
So doing these kind of things, there were a lot of different
mutations that were found that resulted in death at high
temperature.
And then some other tests were done to see if the problem was
that they didn't have the chromosome,
et cetera, et cetera.
OK, and so then in this assay, this assay is actually
something that sometimes we call a complementation assay.
So if you complement something, you're finding the
one missing thing in the process.
And if you have something that's really complex that
might need 10 different things to work in there, the way that
you can get around this problem of how do you find
those 10 is--
so they're going to redo this assay.
We're going to put in this test tube the same things we
did before.
We're going to put in a chromosomal template, and
those dNTPs, and the co-factors, and then the
buffer, and the radioactive ATP so we can watch it.
But then, what we're going to do is take this extract, and
the extract from the mutant--
so say there's 10 things that we need for this-- the extract
from the mutant is going to have nine of those things
normally, but one of those things is
not going to be there.
One of those things is going to be mutated.
So if we add this mix--
so say this is a problem in polymerase III.
We don't know this yet, but.
If we were going to put the extract in here, that extract
would contain all of those other things that we need for
the process.
That's going to contain the ligase and the polymerase I,
and the topoisomerase and primase and helicase.
It's just going to be missing one thing.
So then when you do a fraction, and you fractionate
the wild type, and you're able to find one protein at a time
to add back, then we might be able to find that one missing
enzyme, the one missing DNA polymerase III, in this mix.
If there were 10 things that you needed, there's no way you
could isolate magically those 10 things.
You have to do it one at a time.
OK, so this assay is actually kind of at the heart of a lot
of biochemistry and a lot of genetics, where you're looking
for this process that can be very, very complex--
like building an eye.
That's a really complex process.
Geneticists will break that in one place, and then try to
find the one thing that's required.
And if you're a geneticist, you're working
with a living fly.
So the fly has already provided all the other things,
except for the one mutant that you have to put in there.
If you're a biochemist, you're in a test tube, you're trying
to provide almost everything you need for the assay, except
for the one thing that you're going to be able to find.
So this complementation concept, it's a really
important thing.
And so for biochemists, it's sort of, what is the one
single protein or molecule that we
need to fix this process?
So what we were looking at is we had a double-stranded DNA.
We wanted to make new DNA.
This is a process that requires many things, so we
have to simplify it so that we can just find
one thing at a time.
For geneticists, the idea of complementation is also kind
of the same thing, but you've seen it in a different form.
And again, it's our two mutants, two things
contributing to the same process.
So one of the ways that you actually saw complementation
in action, even though we didn't call it that--
so let's just say, normally, flies have red eyes.
And if we have a mutant, mutant 1 had white eyes, and
mutant 2 had white eyes.
And if we cross mutant 1 that was white by mutant 2 that was
white, and we got red, that's something we talked about.
Before, we said, OK, that means that
there's two genes, right?
But what it also means is that each one of those genes could
be contributing to making red.
If we have one of those genes working, then the other one
isn't needed anymore.
So in this case, so mut 1 isn't a gene that's required
to make red.
I should also back up and say something, that this works
when the mutations are recessive, which would be true
if we got this kind of result.
So they're both in genes that are required to make red, but
they're not in the same gene.
So we talk about this is being complementing.
The process is the ability to make red.
Both of these have problems in it, but
when you combine them--
so if we think about that on the chromosome, so it doesn't
have any of whatever 1 is.
But when we look at what it becomes, in both of these
cases, there's some wild type copy of this.
And so, this kind of concept that you add these two things
together, and yet the process gets fixed.
This is something called complementation.
So complementation is something we're actually going
to come back to later in November.
I just wanted to kind of give you an idea-- just tell you
how that word gets used.
And then we'll talk about it again when we try to figure
out how genes are working.
But complementation is sort of this idea of fixing a process
by adding one thing back if you're a biochemist, or seeing
that two genes are contributing to it if you're
geneticist.
OK, that probably didn't actually clarify
matters any, did it?
You're all silent.
OK, so you guys can ask me this after
class, or ask in meetings.
In terms of complementation, this is not part
of your first midterm.
I just kind of wanted to go back and say for that
experiment that I explained the first time, a little bit
more about the different pieces of it.
So one of the other things that came up was when I talked
about making mistakes.
So when you're replicating the DNA, there's a lot of
different things going on.
A lot of things have to be done right.
And it's very, very important to make sure that, in the end,
the new DNA is an accurate copy of the old.
And so, a great question came up, which was--
I said that there's a way, that there's a little thing
that slides along and looks for mistakes, looks for when
bases aren't matched up correctly.
And Haroon asked this question, well, how do you
know which--
if there's a mistake, if they don't match, how do you know
which one to get rid of?
How do you know which one to fix?
And so he actually looked it up, and is going to explain
some of it to you.
This takes an enormous amount of guts to do this, so.
OK.
You want me to bring up this?
Yes, please.
Can I write on this?
Yeah.
You can write wherever you want.
OK, so hello, everyone.
I'm Haroon I'm part of your class.
I'm a sophomore.
Some of you might know me.
And I asked a question in class, perhaps the last
question I'm going to ask this year.
And so, my question was, how does it know which one is the
incorrect one?
So the first assumption we make is that the parent's
strand will be correct because if it was incorrect like
overly, or have died already.
So we know it is still working.
So the question still remains, how does it differentiate
between the parent's strand and the daughter strand?
So first thing is, as these cells' life progresses, it's
kind of altered by many things.
And one alteration is that this methylate--
You can use this, too, if you want.
Either way.
That one's just for pointing up there if you want.
Or you can write on there.
So one common error is that the cytosine is methylated, so
you can see right here.
The CH3 groups and the dotted diagrams of CH3 So the
question remains, how does this help us?
And so, when the strand is formed, of course, it's
doesn't have any stuff [INAUDIBLE].
Yeah.
You can just go like this.
OK, so what happens is there's this one protein that kind of
goes along strands, looking for any deformities, any place
where it's either too large or too small.
And once it finds such a place, it recruits another
protein called MutH.
And MutH is specific to a specific strand, and one of
the cytosines in that strand has to be methylate.
So it goes to that strand.
It attaches to it.
And the key here is that the mutation does not affect the
strands which it patches.
It affects the other strand, which would be
the daughter strand.
So MutH is a mild exonuclease, or basically something that
cuts up DNA.
So MutH and the other proteins bind along the DNA.
They make other spots, recruit another exonuclease, which
basically cuts up and dissolves those other parts of
DNA that have been cut up.
And then polymerase 3 comes in, resizes the DNA correctly,
hopefully, and ligase seals it up.
If you have any more questions, you can feel free
to talk to me after class, or you can look it up yourself.
Just uh, mis-match repair, as you can tell, is very
complicated, so this is just like a brief, small
down-to-earth version of it.
OK.
[APPLAUSE]
That was great.
So yeah, so this is exactly what he was showing you.
Those proteins that slide around and recognize that
there's a mistake, and then they recruit this other one
that can tell which side is
Methylated.
It doesn't touch that one.
It makes a hole in the other one.
And then Haroon originally was going to explain all of this
to you, and I suggested that maybe not.
Quite detailed.
But then it's able to peel away this strand, basically.
And then DNA polymerase I usually will actually come in
and fix that.
So it's a cool way of assuming that the mistake is on the new
strand, which is a pretty good assumption, because this is
about copying.
And you'll be able to figure out which strand to fix.
OK, and then one more thing.
So again waking, up this morning--
yet another Stanford Nobel Prize, right?
We're going a little not tired of this yet, but I don't know
what I'm going to do next year.
I feel like if they're two this year, then we're going to
be in a slump next year.
But just kind of a shout-out for all of you that are
interested in computational biology, and I
met a few on Monday.
Where this is actually in chemistry, the Nobel Prize in
Chemistry, but it's really for using computation to figure
out how biology works, how DNA and RNA and proteins work.
So it's another pretty exciting day
out here on the farm.
OK, so back into what we were going to start with today, and
that has to do with mutations.
And so on Monday, I told you a little bit.
So we talked about those small changes where DNA polymerase
makes a mistake, and how to fix it.
And those big changes being things like getting extra
chromosomes or losing chromosomes.
And then this is sort of what I ended on.
I'm talking about somatic mutations--
mutations that happen in things like your skin or your
liver-- your meiotic dividing cells, which might affect you,
but aren't going to be passed down to your children, whereas
germline mutations are things that are affecting
the *** and egg.
You might be fine, but your offspring won't be.
Again, now I just added in that conditional mutation.
All right, so let's look at some of these
big mutation issues.
So aneuploidy is a term--
it's kind of a generic term that means that you have an
abnormal number of chromosomes.
So if you think about humans, humans have 46 chromosomes.
So there are 23 pairs.
And any time you have a different number than 46--
so if you have 45 or 47 or 52--
those are all aneuploid.
They don't have the right number of chromosomes.
And the ones that are relevant for medical things are often
these things called monosomic or trisomic.
And so what these mean is that you're missing one out of a
pair, but all the other chromosomes are normal.
So some human that's monosomic for something is going to have
45 chromosomes.
They're not missing a whole set from each one, but just
one of those.
And trisomic is going to have 47.
And if you think about that, make sure you distinguish that
between diploid or triploid.
So diploid is what we are normally, with 46.
Something that's triploid would actually have an extra
one of every single one of those chromosome pairs, so
that would be 69 chromosomes, OK?
So that's just some terms for these guys.
And if you look at humans, it turns out that having two
chromosomes--
to being diploid, being uploid--
is really important.
So if you look at human karyotype, and you look at
what kind of--
I mean, there are people that have different numbers of
chromosomes, but it's very, very limited.
So what we see among people that survive--
one, we never see humans come to term.
so there are no live births of monosomics.
So monosomics are one missing a chromosome.
They don't survive.
They die in utero.
Trisomics--
and you can kind of makes sense of that.
If you're missing a chromosome, and if you think
about the alleles that might be on different chromosomes,
perhaps you're missing some of the
important functional alleles.
But having an extra chromosome is also bad.
And
so that's a little bit hard to think about that you're
missing particular information of an allele.
It's more about all of those genes on that chromosome, we
have too high of a dosage of those.
So triploids--
sorry, not triploids.
Trisomics.
OK, so triploids would be dead.
So trisomics, we really just have trisomy 21, and
occasionally trisomy 18.
So trisomy 18, usually they die in infancy.
And people with trisomy 21, or Down's Syndrome, can live to
adulthood, but there are a number of heart problems,
mental retardation, stature problems.
They're very clearly affected.
And one of the things that you might notice is the only two
that humans can survive with having an extra chromosome of,
they're the pretty small ones.
OK, so you never see someone who is trisomic
for chromosome 1.
So something about the number of chromosomes seems to be
very important, having that balance of having two and only
two for each one of the chromosomes sets.
But if you think about that, there is one very glaring
exception to this idea that having a set of
two is really important.
And that comes right around here.
OK, so half of you in this room have a very nice set of
these large X chromosomes.
The other half of you in this room are missing one of those
X chromosomes, and you have this puny little Y chromosome
instead, which frankly, does not compensate for all the
things on the X chromosome--
in purely genetic terms.
So why is that allowed?
Right, clearly people with XY chromosomes live happy,
healthy lives.
They have offspring.
So there's something special about what happens with the X
and the Y chromosomes.
So if we said that having two chromosomes, or somehow the
dosage of chromosomes--
not having one, not having three, having two-- if that's
important, then somehow or another, we have to make sure
that the male X chromosome is somehow equal to the two
female X chromosomes.
Somehow, we have to make those two things equivalent.
So we do we do this?
We we've got our females that have two X's, and our males
that have one X. We'll just look at the X's.
Somehow, these guys have to be equal.
And so, how do you do that?
You could make a really active X, right?
So you could say, OK, well, our male X is just going to be
really, really active.
And so, that's going to be kind of equivalent to having
two female X's, because this is a really,
really good X, right?
So that would make them equal.
Or you could say, OK, well, the male X isn't really all
that special, but maybe the female X's have to be
diminished, so they'll be very interactive.
So we have two little, tiny female X's.
So maybe you make less active females.
Or maybe you say, well, we have these
two X's for the female.
Let's just use only one of them.
So that would also be making it less active, but in a
slightly different way.
So that would be saying, OK, we'll keep one active, and
then one of them we'll make inactive.
So it turns out, if you look in nature, all for all of
these are used.
So all of these are strategies that different animals use to
make the X of the males and two X's of the females
equivalent to one another.
So we'll talk a little bit about what happens in humans.
So one thing is just to look at patients.
So humans that come through, what are you allowed to have
in terms of X's?
And it's actually pretty amazing.
So if we look at this, and we say, OK, so the standard male
is XY, the standard female is XX.
But there are women-- they're called Turner, after the
person who described this--
Turner females that have only one X. They don't have another
X, they don't have a Y, but there are alive, and they're
actually fairly normal.
There are a few clinical symptoms--
and most the time, all of the people on here that aren't the
normal male and female have fertility problems.
But for those humans, having two X's and a
Y makes you a male.
This is a another person who described this condition.
And in general, they're male.
And super females having three X's, four X's, et cetera, and
males having the same things with the Y.
So you learn two things from this.
One is that we're remarkably tolerant of how many X
chromosomes we have.
And that the other thing is that whenever you see a Y
chromosome in humans, that is a signal that they are male.
So it was a little bit different than the fruit flies
I showed you a while ago, where in fruit flies, it's
really counting the number of X's, and that
makes you male or female.
In humans, it is really the presence of the Y. If you have
a Y, you'll develop primarily as male.
If you don't have a Y, you'll develop as female.
All right, so we see this, so we have to
wonder, how does it happen?
How is it that we get all these extra X chromosomes?
And then, since these people are not dead, how have they
dealt with it?
What do they do to those X chromosomes so they don't have
to high a dose of them?
So go back to meiosis.
Remember meiosis?
I did this drawing before.
These are fruit fly chromosomes.
Same thing sort of happens where you can get those extra
X chromosomes if you have problems in meiosis.
And specifically, those problems are non-disjunction.
So those chromosomes fail to separate, and you'll end up
having problems with what gets inherited.
So if this is a cell that's undergoing meiosis I, then
you're going to end up creating from here two
different cells that'll go through meiosis, too.
This one's going to be abnormal.
So each one of these will then go through meiosis, too, and
create two more cells.
So all of these are abnormal.
So they're missing their chromosome.
These are also going to be abnormal, having an extra
chromosome.
And so from this, you end up having four defective gametes,
the gametes being the *** and the egg.
OK, so you can have a problem in meiosis I. You could have a
problem in meiosis II.
So you could have meiosis I go perfectly normal, and that'll
end up having one of those daughters
creating two normal gametes.
But then in meiosis II, when the homologues need to
separate, again, they could have this
non-junction problem.
The two sister chromatids could fail to separate.
And so, you'll end up, again, with this one having an extra
X chromosome, and this one missing one.
And you can have problems in both of those, right?
So when you talk about males that have four X chromosomes
and one Y chromosome, we think about where that comes from.
So how do you get four X chromosomes?
You can get four X chromosomes if you had non-disjunction
problems here, so you got both of the X's, and then they
failed again.
And so in the end, this is going to create-- so say this
was an egg--
an egg that has four X chromosomes.
And if you fertilize that with a normal male, you could
create offspring that have that genotype.
Now it's affecting just this one chromosome, as
I've drawn it here.
The other chromosomes are normal.
If there was non-disjunction with any other
chromosome in humans--
the exception of chromosome 21--
we wouldn't get a viable baby, right?
So this process of meiosis could happen with any
chromosome.
Just that in the end, this is the only one that creates
something that actually lives.
OK, so that's how you could get them, but then that
doesn't really solve the problem of, why are you OK
with having four X's and a Y?
And so the answer to this was figured out by Mary Lyon, who
took some observations about what happened when you look at
cells from humans that are XY, or males, XX, females, and
then those exceptional individuals that have extra
chromosomes?
And what she noticed, and what others had noticed, was that
if you look at those chromosome squashes I've been
showing you where you can see all the different chromosomes,
if we just looked at what the X chromosome looked like, in
males, you'll see a nice X chromosome.
In females, you'll see a nice X chromosome, but then you
don't see the other one.
You actually just see this little thing.
And if you look at one of his exceptional females, you'll
see two X chromosomes--
or, sorry.
You won't see two X chromosomes.
Where's the eraser?
Go away.
Back.
How do I make you go away?
OK, this is a mutant that is unable to do this correctly.
OK, so I'm afraid to touch something.
How do I--
OK, here, we solved this.
OK, so bad microscope slide.
Can't see what's on that side, but magically, all the
chromosomes are on the other side of the
bad microscope slide.
And what you saw instead were two of these things.
And these little things--
I guess Barr is the one that found them--
are called Barr bodies.
And just an observation that there were these little things
in there, and that the other observation was that the
number of Barr bodies in the cell was one less than the
total number of X chromosomes.
And so, in an XY, there were no Barr bodies.
So one minus one.
In a normal female, there was only one of them.
And in one of these with three, three X's
had two Barr bodies.
So that made Mary Lyon think, well, perhaps what's happening
to those X chromosomes is that one of them is a nice X
chromosome that's going to be active, but the other one is
going to be compressed into something
that's basically inactive.
It's not going to be looked at.
And that's the way our female X's could be
similar to the male X's.
It's as if there's only one X chromosome, because the other
one, it's not gone, but it's wrapped up in proteins in such
a way that it's basically inactive.
So her idea is that if you take a female--
so this is going to be something that, if you're a
male, you don't have to inactivate your X. We're only
going to look at females.
But if you're female, you have two X chromosomes.
So when you're a zygote, that's the product of the
*** and egg.
One of your chromosomes is from your
father, so the paternal.
The other one's from your mother, the material.
So when you're a zygote, you have both of those.
But at some point when you're in embryo, those cells
randomly start to take one of those X's, and make it into
one of these little Barr bodies.
So if you looked at a female embryo, if you could look into
each one of those cells, you'd really only see one X.
OK, but isn't it always the same X that's expressed, the
other X of the Barr bodies, or does it ever end?
So this is a great question.
So which one is it?
Well, based on some of the genetics, it looks like it's
random which X gets activated.
So if you look at this female, some of her cells would have
been the paternal X there, and the maternal one crumpled up,
whereas other ones would have the maternal X. All of these
cells would have an X. I'm just showing
it in some of them.
So which one of those X chromosomes you actually use,
and which one of them is crumpled into a ball is going
to be random.
And that's going to happen sometime in the embryo.
And then when you grow up, and become--
now you're a baby lying on your back.
That's how you can tell it's a baby and not an adult.
It's lying on its back--
is that this infant is going to have some of her cells.
So some of the cells have the active X chromosome from the
father, and some--
some have the maternal, some have the paternal.
OK, so that's kind of a bizarre initial idea.
But there was some evidence, and one of the most striking
examples is what you can see in something like a tortoise
shell cat, where based on the behavior certain X-linked
traits, this kind of idea wasn't such a bad idea.
So let me tell you the genetics of this.
So this is a tortoise shell cat.
It's got orange and black.
There are a couple of things known about
tortoise shell cats.
One is almost all the time, they're females, OK?
So it's very, very rare to see a male tortoise shell cat.
They're almost all females.
And this color--
this orange versus black--
this is a sex-linked trait.
And if you want a tortoise shell cat, the way that you
get this is that you cross a black female cat--
so if you think about her X chromosomes, she's going to
have a big B allele that gives a black color.
And you cross her to a male cat that's orange.
And the little b is for the orange.
But he's a male, so he's got a Y chromosome.
So if you look at the project of this, if you look at the
males, they all have an X from their mom with a big B, so
they're all black.
But the females are heterozygous.
OK, so they have one big B, and one little b.
So if these two things were completely dominant one over
the other, you'd expect this one to be
black or perhaps orange.
But actually, you don't see that.
You see this kind of mixing.
And that's because these two get inactivated
So sometimes, it activates the big B X. And in those cells,
it produces orange.
In other times, it's inactivating the little b, and
those produce black.
So what you end up having are-- so here, notice a nice
black color.
If we were to look at those, the big B X is active, and the
other one is a Barr body.
But if you looked at the orange ones, that
would be the opposite.
So the little b X would be expressed, and the other one
is a Barr body, OK?
So once in awhile, you'll see a male that is a
tortoise shell cat.
Any idea of how you get a male that was a tortoise shell?
Klinefelter's.
Yeah, Klinefelter's.
I don't know if they call them Klinefelter cats, but exactly
that same--
yeah, exactly.
Yeah, so any time something has more than one X
chromosome, it could be inactivating it.
So the only times that you have males that are tortoise
shells are the XXY's.
OK, so what about this one?
Any idea how you get that one?
So calico cats are basically this one.
They're the tortoise shell cat.
That's how you get the black and the orange.
But then on top of them--
remember when we talked about epistasis?
There's another gene that controls whether or not you
make color at all.
And that gene is actually an autosomal gene, and there's
some other complications of that.
But there's also another gene in this mix where in some
cells, you don't make any color at all because you're
inactive for this color piebald gene.
Anyway, that's just an illustration of how genetics
gets really complicated.
All right, so that's kind of a cool demonstration of how you
can equalize different X's.
Yeah?
You mentioned that in the embryonic stage, the Barr
bodies start to be formed.
Once a Barr body is formed in its cell, does everyone get a
daughter cell?
Like through mitosis, remain a Barr body for that specific--
Yeah.
That's a great observation--
or question, and observation, in a way.
So his question is, if you're a cell, and you have decided
that you're going to have the maternal X active and the
other one not, what happens through mitosis to the two
daughter cells?
Are they going to look just like the mother cell with the
maternal X?
Or are they going to have the paternal one?
And so, there is a possibility that they switch.
But most of the time, they actually keep
the same one repressed.
And that's why you actually get patches.
So you'll get a patch of one skin or hair founder cell
that's black, and all of its progeny that are near it,
they'll all be black because they've inactivated the same
X.
So it's not 100% of the time.
So clearly, just from looking at phenotypes, it can switch.
But most of the time, you inactive the same
one over and over.
Yeah?
OK, so how come with some traits, you have women who are
carriers of-- like they don't express it, but they can pass
it on to their children.
Because with this one, like any of these like
heterozygotes have a co-dominance sort of thing
rather than being a carrier.
Right.
So he's asking a really, really--
it's a tricky and sophisticated question of
saying, OK, so we've been talking about
codominance or dominance.
How do you deal with that if you're talking about an X
chromosome where half the time, one of those X's is just
not going to be active?
So it turns out for most of the things-- and this is just
purely just based on all the evidence that we have--
it it's a random enough process that having one or the
other inactivated means that if you look at the thing as a
whole, it's fine.
And so as an example, say it's whether you have a mutation
that doesn't make a liver enzyme.
OK, so on your X. If you look in your liver, they're going
to be enough cells that have the active one, and enough
that don't have the active one that, together, your liver is
going to function fine.
So it's kind of a-- if you think about the whole
population, you're talking about a million cells.
And usually half a million cells with the right--
so the active one will still work.
So for a lot of phenotypes, it turns out that having that X
inactivated half the time is actually--
overall, it's still fine.
This happens to be an exception where if you have
that X inactivated, you simply don't make these colors.
This is a nice illustration, but it's
probably less than 10%--
maybe even less than 1%-- of the time do our genes actually
behave this way.
Yeah?
Quick question.
How does it choose which X to connect with.
I know it's random, but--
So how do you choose?
I don't know.
At some point in the cell, some machinery comes over,
grabs a hold of one of the chromosomes, and says, you,
and you're going to get crumpled up.
I don't know how that machinery is targeted to any
particular x.
It might just be that it starts to build on both, and
whoever gets there first gets completed, and
the other one doesn't.
So I don't know how they choose which X to inactivate.
I mean, you kind of hope, like on one hand, it would be
really nice if we could fix ourselves.
So say you had an X chromosome from your father--
or if I had an X chromosome from my father, you wouldn't--
and it had some very deleterious allele on it,
something that could potentially cause disease. it
would be great if I could always inactivate that one,
and not inactivate the other one.
But so far, we don't have any evidence for that.
Yeah?
So how does the [INAUDIBLE]?
Yeah. that's a great question.
So what happens to Barr bodies in meiosis?
So a lot of things get reset when you go from making your
somatic cells-- most of the cells in your body-- to
starting to make the ones from your germ cells.
So not the actual cells that go through meiosis, but one
step before those, the ones that make the cells that can
go through meiosis--
they reset a lot of things.
And so, when people talk about epigenetics, those are
epigenetically reprogrammed.
The Barr body, it's called heterochromatic, and
heterochromatic is a very vague definition which
essentially says that the chromatin--
which is all these proteins, wrap it up, and so it's
inaccessible--
so chromatin changes.
So heterochromatic regions become unpacked.
So there's a lot of things that happen
just at that stage.
So the Barr bodies at that point are going to be
unwrapped, and you're going to have an X chromosome.
So for a gene like color-blindness, they say that
males are more prone to being color-blind because they only
have one X chromosome.
So if the female--
it's heterozygous for that gene, the female half of the
Barr bodies are expressing their color-blind allele.
That female's still going to be able to
distinguish colors correctly?
Yeah, so again, because there are enough neurons--
and I can't remember, rods or cone.
The cones, I guess--
enough cones in your eye, because there's a million of
those, if some of them are not very effective,
you're usually OK.
It would be actually interesting if someone took a
really, really fine-scale color-blind test.
So usually, it's like, can you see this red number nine in a
bunch of green dots?
But perhaps if you did a very, very sensitive one, you'd
start to see a difference.
But generally speaking, you don't see a difference because
there's enough.
OK, cool.
All right, so conclusions from that are that it is important
to have the right number of chromosomes, but there are
some really interesting mechanisms that make the
dosage between males and females equivalent.
So the one in humans where we inactivate something--
I also like in fruit flies, you actually make the male act
super active.
So that's kind of an interesting way of boosting
the male X, and just sort of leaving the
female ones the same.
OK, so that was kind of an illustration of really, really
big mutations--
mutations where you're missing an entire chromosome, or you
have an additional entire chromosome, or ways of dealing
with having extra chromosomes.
But most of the time, mutations are
not quite that big.
They're smaller.
So they're parts of chromosomes, or as we talked
about with DNA polymerase, it can make mistakes, and that's
a very small mutation.
But we're going to talk about a couple of different
chromosomal rearrangements.
And these are things that do happen in us.
And they happen in fruit flies, as well, and they're
kind of useful there.
So you can have not missing an entire chromosome R--
not missing the entire chromosome, but
missing parts of it.
And so if you're missing parts of it,
that's called a deletion.
So you're missing information.
Sometimes it's called a deficiency.
So you might see that in a book.
An insertion, as you might guess, is extra information.
So sometimes, this is duplicated.
And I'll show you an example.
Duplicated from the same chromosome, or who knows where
it's inserted from.
An inversion is that part of your chromosome has actually
just been flipped.
So you might start over here, if you think about a linear
molecule going from here to here, the middle part of it's
just been flipped around.
So often, this has the same information, but in a
different order.
And a translocation is where part of one chromosome
actually ends up on a part of a different chromosome.
OK, so I find these much easier to
think about just visually.
So let's just say we have a chromosome.
I'm always drawing them like this.
I'll actually show you a picture about why I
draw them like this.
But let's imagine there's some genes on this chromosome.
So we'll just designate these regions on here.
So if you have a deletion, you're just missing some
information.
So as I've drawn it here, we'd say, this is a chromosome, so
this is a deletion.
So a deletion, or little delta, of C and D. So it's
just missing from there.
And insertion would be a bit of the opposite.
So you'd have, in this case, the same region twice.
OK, so I think these are pretty straightforward to
understand at least what we're calling an
insertion and a deletion.
So how do we know some of these things?
So a lot of this work was done in these things called
polytene chromosomes.
And they're actually from the salivary
glands of fruit flies.
And there's a step where the cells aren't dividing, but
they're actually still making more and more of the
chromosomes.
And so there's actually 1,000 chromosomes all kind of packed
together, so it's much easier to see in a regular
microscope.
And so what this is is actually, this is pair of
chromosomes where this fruit fly is homozygous for almost
everything.
So it's very hard to see--
they're at a stage where the chromosomes, the homologues
are lining up.
So chromosomes would be lined up in prophase or
metaphase of meiosis.
And if they're homozygous, it almost looks like one big wide
chromosome.
But if they're heterozygous for a lot of things, you can
see these two chromosomes lined up, but they're not
always the same.
So in many places, those bands are the same.
But in other places, they're not quite the same.
So you can see here, it's missing some of these dark
bands, and there are some there.
OK, so you could actually see deletions or assertions by
whether or not the bands are here in these chromosomes.
On human chromosomes, it's extremely
difficult to see them.
But in fruit flies, it's been easier.
All right, so inversions are basically flipping
information.
So if we had an inversion of it, we'd end up getting E over
here, and D and C and F. So what do you think?
Inversion--
we still have all the same information.
Think that's going to be fine?
Any--
could be.
I guess it could be if it's in the middle of a gene, or
between two genes.
Yeah.
So they're called break points.
So as I've drawn them here, it looks like everything is fine.
But at some point, something happened over
here and over here.
And what is the point where it got switched is in the middle
of the gene.
You may actually disrupt that gene, and
that can be a problem.
And the other thing that happens in these pretty
commonly is that a gene--
so a gene that's going to make a protein
gets moved to a place.
So there might be a gene that normally only makes a protein,
say, in your liver.
But it gets moved next to another gene, another gene
that makes something that's in all cells all the time.
And so this one gene that normally has the information
near it, telling it only be made in the liver, now is
sitting next to some information saying, every cell
needs this.
And so that gene is going to be made into many cells, and
that can be a problem.
OK, the inversion of where those break points are can
make a difference.
And then, in translocation, you actually saw these already
when I talked about Barbara McClintock's maize, the way
that she had these little knobs on the end of the
chromosomes.
So what that little *** on the
chromosome was is that actually--
so this was the chromosome--
there's actually a chunk of this other chromosome that was
put on the end.
OK, so those are some major chromosomal problems.
How do you get them?
And there's really two ways that you get them.
So one is if you walk into a nuclear reactor without any
protection, you can damage these.
So these can be damaged by pretty bad radiation.
And so if you have a chromosome and you break it,
this is this all-out warning to the cell.
So if a cell recognizes that there's a broken chromosome,
recognizes those ends, it will shut everything down, and the
main thing it tries to do is fix that.
That's seen as one of the worst problems that
the cell can have.
And so it will try to fix this, but it doesn't always
fix it perfectly.
So if there's a big chunk missing, it's going to join
these ends together, but you're going to be missing
some information.
And that's how you end up getting a deletion.
But equally problematic is that cells have other ways of
error-correcting other than what Haroon told you about
before where they see a break, and they try to fix it by
adding new stuff in there.
And they can add the wrong new stuff in there, and so you end
up getting insertions.
And the other thing is that this end-- this sort of broken
chromosome end--
it really wants to reattach to something else.
So if it doesn't find itself, it's going to go find some
other chromosome, and just attach to that.
So this one can end up finding some other perfectly happy
chromosome over here, and just saying, OK, I need an end.
I'm going to join you over here.
So that's radiation ways that this can happen.
I think the more interesting ones are the meiosis ways,
because that actually is something
that comes up before.
When we talked about meiosis, and saying that the homologues
have to line up, it's really pretty amazing.
They have to line up perfectly.
So we have--
let's see, we have 23 pairs of chromosomes.
We have--
people think about 30,000 genes, so you've got 1,000 or
so genes on a chromosome.
Is that right?
Yeah.
And you have to make sure that when you have recombination
that those genes--
the recombination has to be precise to a single
nucleotide of DNA.
Because if you're off in recombination, then you're
going to screw up all of those genes.
And so, normally when we have meiosis-- so imagine you've
got these two chromosomes, this homologous pair.
I'm just going to draw a little bit in the middle.
And we've got the, say, recessive alleles over here.
So if you have recombination happening--
you're all mapping stuff right now, so you're pretty keen on
recombination, I'm sure--
then we want to make sure that what you get out of this after
recombination is a chromosome.
So here, we have a big B, big C, little d, little e; and in
another chromosome, little c, little b, little
c, big D, big E--
that they've recombined, but they still have an allele of
each, of B,C, D, and E. They have all that information.
But what if they're off by a little bit?
Right, so what if we take those same ones and say, we've
got B, C, D, and E. And so what if we
don't align them perfectly?
Right, and think about what happens then.
We're going to get a chromosome from here that
looks like a big b, and then a little d, and a little e.
And then on the other side, a little b, a little c, a big C,
a big D, and a big E. So if they're not lined up
perfectly, you actually end up getting chromosomes that have,
in this case, a deletion-- right, we're missing some
information--
and this way, an insertion.
OK, and that's a real problem.
And that's one of the major things that in meiosis, they
can spend a lot of time in prophase lining up those
chromosomes to make sure that the recombination is in
pinpoint accuracy.
And these translocations--
these happen because, again, chromosomes--
so we saw a little bit of this with the X and the Y
chromosomes.
So those are different chromosomes, but there's a
region of those that let's them pair.
There's a region of those that's similar enough that
they actually get together.
So that's what should happen.
But occasionally on other chromosomes, the regions that
are similar enough to regions on-- so chromosome six might
have a region that's similar enough to chromosome five that
they actually try to pair, and then you get problems.
So you'll have some region that looks similar enough,
say, here and here that they'll try to pair, and try
to do recombination.
And then as you can imagine from this one, you'd end up
with a chromosome that's got a little bit of purple on here
connected to the black one, and then a little bit of black
connected to the purple one, whereas the other ones
wouldn't be participating in this.
And so, those will also lead to problems.
And I'll show you in a moment, actually, some weird things
that can happen when you have those.
OK, so some consequences--
so with these arrangements when you're losing or gaining
information, as I've shown you there in meiosis, that cell
might actually be OK, because it has all of the information.
But when it tries to pass down one of those homologues to the
offspring, they're not going to have the
right number anymore.
There's also problems when these try to pair.
So if you think about this, if they're trying to line up, and
one of them is missing some information--
so if you have a deletion where you're missing this bit
in the middle, if you have a wild type chromosome, and
they're going to try to pair, there's going to be a region
in here where they can't pair, because there's
nothing that's similar.
So what's going to happen is that recombination won't be
able to take place in here.
It's just not going to be possible.
Same thing with an insertion.
The insertion would also not be able to pair.
And then if you have these translocations--
so if you imagine, say you've got something where you've got
a bit of chromosome 4 and a bit of chromosome 5 together,
you get into these really weird situations where in
meiosis, you actually get things with four chromosomes
trying to pair with one another.
So one of them will be the normal chromosome 4, one of
them will be the normal chromosome 5, and they'll be
trying to pair with these sort of hybrid chromosomes.
So these are the two translocated ones.
And the other chromosomes are really confused.
They're trying to figure out, well, who do I pair with?
So they end up with this little thing.
And then your cell's like, wait a minute, I know how to
take two homologues and separate those.
I know how to take two sister chromatids and separate those.
I have no idea what to do when I have this
little star-shaped thing.
And so usually what happens is this little thing gets sent.
All of this goes to one of the daughter cells.
And so, then you end up compounding things, and
getting even worse, because now one of them is missing all
of the 4's and all of the 5's, and the other one has all the
extra ones.
All right, so in terms of what you should know about these
types of mutations, you don't need to know all of the
details of-- it's very medically important, but we
can't really get too much to the mechanism of these.
But what I think you should be familiar with is how problems
in meiosis can lead to those different kinds of insertions
or deletions or translocations.
All right, so I've kind of said all of the reasons that
these mutations, these big mutations, are bad.
But actually, they can be pretty useful.
And so, what we'll really conclude with on Friday is a
little bit about how these are useful--
so how they help us figure out where a gene is on a
chromosome.
And then, how they also help us figure out what a gene
actually does.
OK, so that's going to be on Friday.