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Let's dive in
today and look at

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how geneticists use genetics.
I've told you up until now about

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some of the history of genetics
and how it gave rise to our

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understandings about genetic
transmission in traits, about

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genetic mapping,
linkage analysis,

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how all this helped confirm
the Chromosomes Theory.

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And we wove in a number of concepts
about how scientific theories are

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developed and data is interpreted
and intuitions are made,

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and then how they're actually proven,
what sort of evidence it takes to

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actually achieve
conscientious around theories.

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And that often takes sometimes
years, many times decades before

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full contentious is achieved around
things. Today I want to turn a

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little bit to the experimental uses
of genetics in a more day-to-day

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fashion. And you will recall this
coat of arms that I put up here.

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Function. Gene.
Protein. Biochemistry.

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Genetics. And I told you about how
these were two different ways to

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study biological function.
Today I want to talk a little bit

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about how we use genetics
to study biological function.

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And, in particular, I'm going to
pick some examples of how we use

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genetics to study biological
function that have to do with the

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biological functions
of biochemistry.

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So already we're beginning to look
ahead to this connection between

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gene and protein, which
molecular biology will

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establish for us. So, suppose
you want to do genetics.

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You've got to study some organism.
We talked already about Mendel's

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choice of organism, the pea.
We talked about some of its

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advantages and disadvantages.
Advantages you could get pure

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breeding strains in the market,
you could, when you're done with the

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experiments, feed it to the other
monks. There were a lot of things

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like that, that were
advantageous about the pea,

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but it had problems of generation
time. You would only get,

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certainly in Europe, a generation or
so a year. In Northern Europe maybe

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you could squeeze a second
generation in not so good.

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Fruit flies, a very attractive
system in many respects because you

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could grow many,
much larger numbers.

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The generation time is on the order
of two weeks or so to go from a

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fertilized fly embryo, a fly
egg developing into a fly,

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developing into a mature adult,
able itself to have offspring.

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So, very attractive. There are
other systems that people studied.

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And, of course, one of the reasons
they study this system is because

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it's interesting, I'm sorry,
because it's tractable.

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And the other reason is
because it's interesting.

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So, tractability is very
important to a geneticist, right?

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The number of whale geneticists
is few, for the most part.

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But we also want to choose our
system because of what it will tell

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us about the system we want to
study. Like if you want to study

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distinctive things about the immune
system, you might want to study them

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in mice, or if you could
even study them in people,

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although you can't set up crosses
in people. We'll come to that on

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Monday. If you wanted to study
things about basic aspects of

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development, you might
study them in fruit flies.

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And if you wanted to study basic
biochemistry, the place to study

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basic biochemistry might best be
done in single-celled organisms,

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which also have to carry out
biochemical pathways like glycolysis

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and synthesis of amino
acids and things like that.

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They're going to be, by far,
the most tractable systems.

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And so, people are particularly
fond for doing things like studying

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basic biochemistry and many other
aspects of basic molecular biology

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to studying the organism yeast.
Yeast is a friend of human beings.

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Certainly, yeast has been an
intensely studied organism because

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of its practical benefits
in the making of bread,

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in the making of beer.
So, fermentation processes,

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dough rising and all that. But
yeast also is a tremendously

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important organism for the
geneticist. It is an extremely

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elegant experimental
system. Yeast is a fungus.

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It is a single-celled eukaryote.
That is true nucleus. It's got

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chromosomes that pair up. It's
cells, through a first order

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approximation, that are
an awful lot like your

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cells in terms of having all of
the basic important eukaryotic

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organelles in the nucleus,
mitochondria, other things like that.

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So, yeast is a
great model for many

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purposes. And we're not going to
talk much about the cell biology of

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yeast, but I do want to talk
about the husbandry of yeast,

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how it is that you grow yeast.
So, the way a geneticists grows

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yeast is take growth medium
that has lots of rich nutrients.

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You could take a broth with lots of
amino acids and all sorts of stuff,

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you know, a little bit of
salt, lots of water of course.

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And if you take a single yeast cell
and it's got lots and lots of rich

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nutrients in this broth here,
you put your yeast cell into the

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broth, so I will do that.
Here's my flask, here's my little

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rod which has a yeast cell or a
couple of yeast cells on the end of

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it. I put it in there and I grow
it at an appropriate temperature.

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Let's say 30 degrees, for example,
would be a nice temperature.

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I could do that if I wanted to.
Then a C obviously. I grow it up

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and I get a culture of yeast in
there. And I can tell because this

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nice clear broth is now all cloudy
with yeast that's grown up in it.

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Now I want to study these guys, so
what I do is I pour them out onto a

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Petri plate. The Petri plate has
on it a medium, a solid medium,

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an agar medium that
again has nutrients.

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And if I pour this out,
and I pour out a lot of it,

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what will happen? Well, there will
be yeast all over the place and it

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will be very smootsie. There
will be like yeast cells

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everywhere and it's
not very organized. So,

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what I want to do is I want to
take that and I want to dilute it.

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I want to take only a little bit
of the broth and put a little bit of

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the broth on my plate. Maybe
I'll have diluted it first.

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And then I want to spread it
around with a little spreader,

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here's a little glass spreader maybe
or something, and push it back and

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forth, so that really there are just
individual single cells scattered

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randomly, scattered around.
And so, then this cell begins to

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grow and divide and divide
and divide and I get a colony.

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A little hill of cells all of which
descend from a single cell that was

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put into that position. And
the reason I know that they all

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descend from a single cell is
because most of this plate does not

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have cells on it. Most
of the plate is sparse.

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I've just got cells, cells, cells,
cells scattered about. And because

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of that I know that these
had of been individual events.

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These things are called colonies.
Now, when yeast grows and divides

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like that, let's take a moment
and talk about its life cycle.

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We'll introduce its life cycle
here. Yeast proper eukaryote,

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so it has a diploid stage. It
grows as a diploid. And it can

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undergo mitosis in which all
of the chromosomes line up, as

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we talked about. They've
already pre-replicated so

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that they'll be ready to divide up
and give one to each daughter cell,

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and there you go. N for yeast is
16. Yeast happens to have 16 pairs of

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chromosomes. Peas had seven.
Humans have 23 pairs. Every

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organism has its own yeast of
16. Now, what we do is we undergo

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meiosis to make haploid cells,
sperm or eggs in the human

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population. Yeast also
undergoes meiosis to make spores.

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It sporulates and it produces
spores. And it turns out these

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spores, of course as you would
expect, have N chromosomes.

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They undergo meiosis just
as we drew it on the board.

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And these can come in two flavors.
They happen to come not in male and

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females, but A and alpha, there
you go. A and alpha cells can

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mate together to produce,
again, a diploid. They fertilize

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and can produce a diploid. They
fuse to do that. And you now

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get back to a diploid from your
haploid. So, this looks just

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identical to the human genetic cycle
here, but there is one difference.

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What's the difference?
Sorry? Time. Yes,

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it's true. Yeast can divide
much more rapidly. Yeast can have

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offspring extremely rapidly
over a course of a day or so.

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And humans take somewhat
longer than that. They,

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for example, have to wait until
they get out of college to be able to

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reproduce mostly. What
else? There's one other

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important thing. It turns
out that yeast can also

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undergo mitosis as a
haploid. In other words,

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the haploid cells of yeast, when
it makes individual haploids,

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they can continue to grow
indefinitely. By contrast,

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your gametes cannot. You do not
have an independent human stage in

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which you are haploid, or
your gametes are haploid.

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Whereas, yeast can hang out as a
haploid for a very long time until

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it decides it wants to mate.
This is very convenient for

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geneticists. Geneticists like this
because it means we can grow the

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thing as a diploid, we can
grow the thing as a haploid.

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When we want to mate them,
we can mate them together,

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but we can also study them alone.
And, you could imagine, this is

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going to be really good for
studying recessive traits,

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right? So, that's one of the
reasons why geneticists are fond of

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yeast. There are many reasons
geneticists are fond of yeast.

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Just growing yeast, it
smells very nice in the lab.

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For example, try growing E.
coli by comparison. So, now,

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it turns out that yeast is very
happy if you grow it on rich medium.

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But yeast can grow on minimal
media with very few macro molecules.

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It needs a carbon source which
is some sugar that it can ferment.

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It needs a nitrogen. It needs
some simple source of nitrogen.

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It needs some simple source of
nitrogen. It needs a source of

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phosphorus. It needs some other
trace salts and things like that.

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And obviously it needs some water.
That's it. If you think about

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what's in a yeast cell, like
it's got phospholipid bilayers.

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But you're not giving
it any phospholipids.

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Why is it able to grow? It
makes them. What about proteins?

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They're made up of 20 amino acids.
You're not giving it any amino

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acids. Why? It makes them.
Yeast is extraordinarily

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self-reliant. You, by contrast,
are not as self-reliant.

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There are a number of amino
acids which, if I don't give you,

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you can't live because you don't
actually have the ability to make

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those amino acids. But yeast
is able to make the vast

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majority of things. Basically,
you almost just needed

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to give it the elements. As
for carbon sources and things

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like that, it's very happy with a
wide variety of fermentable sugars.

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You can give it glucose.
You can give it sucrose.

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You can give it galactose.
You can give it fructose and it

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will deal. So, yeast
is very well set up

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metabolically. So, it's
got all of these pathways

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of the sort Bob has talked about for
being able to breakdown the things

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you give it and being able to
synthesize up the things it needs.

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Now, yeast, of course, is not
stupid. Because if you give it

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amino acids it will use it. If
you give it all sorts of other

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things it will use it. So,
yeast is able to use rich media

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that have lots of complex
nutrients and macromolecules.

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So, it has an ability, it has
everything it needs to make

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these things, but it has
an ability to regulate that.

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So, the processes, the
enzymatic pathways that produce

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complex macromolecules,
amino acids, phospholipids,

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et cetera, will be down regulated,
shut off, or at least decreased if

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you provide it with
these macromolecules.

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That's an interesting question
of how it manages to regulate its

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biochemistry. Why does it care?
Why doesn't it, why not just have

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those pathways be on all the
time? Sorry? Waste of energy.

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It needs ATP. It costs money.
So, at the beginning probably they

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were on all the time,
but some yeast evolves,

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or some precursor to yeast
evolves that's able to regulate it.

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That one is able to be
more frugal with its energy.

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It outgrows its other ones
and then another, dah, dah,

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dah. Any place you can make
a few ATPs here or there,

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eventually the organism that does
it will out compete the organism that

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doesn't. And so, rather
fine control of this,

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which is a topic we'll
come to in a couple of days,

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gene regulation and other
kinds of pathway regulation is

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very important. OK. So,
we want to know how does it

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do it? What are the enzymes?
What are the pathways? How does it

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actually make,
oh, I don't know,

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arginine? How does it make arginine,
amino acid? How would you find out

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how yeast makes arginine? How
can yeast synthesize arginines?

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So, you remember our picture that
the biochemist wants to study a

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problem by grinding up the cell
and purifying a component able to do

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something. So, a biochemist
might want to grind up

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the cell and purify an
enzyme that can make arginine.

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Form what, of course, is
an interesting question?

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And then the thing that made the
thing that was used to substrate,

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et cetera, et cetera.
What would a geneticist do?

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How does a geneticist approach
the problem with how does

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yeast make arginine? Find
a yeast that cannot make it,

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that's what we do. That is.
So, what we need is a mutant.

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A geneticist wants a
yeast that cannot make it.

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A geneticist wants mutants.
How do you find the mutant? You

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find the mutant by
going on a mutant hunt.

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That is what geneticists refer to it
as. And it's a very exciting thing.

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You go off, load up the guns and go
off into the bush on a mutant hunt.

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And so, I want to talk about
the strategy for a mutant hunt.

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How do we look for a yeast
that can't make arginine?

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00:17:16,000 --> 00:17:20,000
Sorry? Cannot. I've got a
yeast that can make arginine,

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00:17:20,000 --> 00:17:24,000
because normal wild type yeast
can grow on minimal media without

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arginine supplied.
And, when I examine it,

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it's got arginine in it.
Yes? So, who should I find?

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Proteins that contain arginine and
then it doesn't have the proteins

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that doesn't have arginine.
Interesting. Now, the problem is

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almost all proteins will have an
arginine, or the vast majority of

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them. And a yeast that lacked all
those proteins that didn't have

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arginine would not be much
of a yeast. I think it would

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be pretty dead. So, it's
a good thought if it was a

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more dispensable function.
But that's going to be tough.

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Or, maybe I can use the fact
that it's dead. Now in a sense,

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can I find the yeast? Yes?
You had a thought on this.

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Yes? Kill all yeast that
make arginine, excellent.

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00:18:20,000 --> 00:18:25,000
So, if I had a chemical agent
that could kill yeast that can make

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arginine, I could only get the yeast
that make it. How would I do that?

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That's a very interesting idea.
You're right. You could construct

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00:18:34,000 --> 00:18:38,000
the chemical molecule in the
arginine pathway which when it was

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broken down enzymatically
made some toxic product,

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and only those yeasts that couldn't
break it down would be able to grow,

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00:18:46,000 --> 00:18:50,000
et cetera, et cetera, and I could
select. That's a very cleaver idea.

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But I'd also have to know an awful
lot about the pathway in advance.

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00:18:54,000 --> 00:18:58,000
So, suppose I didn't' know the
pathway. Suppose I knew nothing

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00:18:58,000 --> 00:19:03,000
about how arginine gets
made. Yes? Excellent. So,

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00:19:03,000 --> 00:19:09,000
I take, I mean geneticists are
simpleminded folks and they like

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00:19:09,000 --> 00:19:15,000
simple solutions. Take
medium in which you've given

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00:19:15,000 --> 00:19:21,000
the yeast arginine, grow it
up, and then pour it out on

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00:19:21,000 --> 00:19:27,000
a plate that doesn't have arginine.
Everybody got this idea? So, we're

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00:19:27,000 --> 00:19:32,000
going to take yeast. We're
going to grow it up in medium

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00:19:32,000 --> 00:19:37,000
which contains arginine
with arginine. So,

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00:19:37,000 --> 00:19:42,000
now yeasts, those mutants that arose
by chance that are unable to make

253
00:19:42,000 --> 00:19:47,000
their own arginine are still able
to grow here. And then we dump it out

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00:19:47,000 --> 00:19:52,000
onto a plate that has minimal
media without arginine,

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00:19:52,000 --> 00:19:57,000
no arginine, and those ones that can
grow up are the ones that we're not

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00:19:57,000 --> 00:20:02,000
interested in. And the
ones that don't appear are

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the ones we're interested in.
But, wait a second, that's the

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00:20:07,000 --> 00:20:12,000
problem, isn't it,
because they're not here.

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00:20:12,000 --> 00:20:17,000
How do we study them if they're not
there? What can we do about that?

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00:20:17,000 --> 00:20:22,000
Yes. You want to see if you can
help us. Remove the ones that grew

261
00:20:22,000 --> 00:20:27,000
up. So, get in there,
scrap them off, now put some

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00:20:27,000 --> 00:20:34,000
arginine on. We're
getting to the idea.

263
00:20:34,000 --> 00:20:42,000
Maybe we can set this up
more elegantly, though.

264
00:20:42,000 --> 00:20:51,000
Thoughts? How can we, yes?
Make a bet? Make a guess?

265
00:20:51,000 --> 00:21:00,000
I can make that guess,
but how do I find them?

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00:21:00,000 --> 00:21:06,000
Here's a simple,
simple, simple idea.

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Let me try a simple idea. How
about I grow up these yeast,

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00:21:13,000 --> 00:21:19,000
and instead of plating
them on minimal medium,

269
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let's be good to them. Let's
plate them on minimal medium.

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00:21:26,000 --> 00:21:32,000
Good. That's interesting. Let's
plate them on minimal medium

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plus arginine. Or,
actually, if we wanted to,

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00:21:38,000 --> 00:21:43,000
we could even plate them on rich
medium. We'll be really good to

273
00:21:43,000 --> 00:21:49,000
them. Either way. So, now,
let's let each one grow up.

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And here will be the ones that
can grow and the ones that can't

275
00:21:55,000 --> 00:22:00,000
grow with arginine. Now
let me take a plate that is

276
00:22:00,000 --> 00:22:04,000
minimal medium. And now
let me take a toothpick,

277
00:22:04,000 --> 00:22:08,000
put a little toothpick there and
carry over this colony to there.

278
00:22:08,000 --> 00:22:13,000
Let me take a toothpick and
carry this guy over to here and a

279
00:22:13,000 --> 00:22:17,000
toothpick and carry this
guy to here, and a toothpick,

280
00:22:17,000 --> 00:22:21,000
and a toothpick, and a toothpick.
And all I have to do is keep

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00:22:21,000 --> 00:22:26,000
transferring, one at
a time, these colonies.

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And now I can see that somewhere
there was a colony that grew fine

283
00:22:29,000 --> 00:22:33,000
when I gave it,
say, rich medium,

284
00:22:33,000 --> 00:22:36,000
or minimal plus arginine, and
a colony that didn't grow when I

285
00:22:36,000 --> 00:22:40,000
put it on minimal medium.
That would at least show,

286
00:22:40,000 --> 00:22:43,000
so, of course, the issue is I first
have to find them by growing them on

287
00:22:43,000 --> 00:22:47,000
something where I've given the
arginine and then I can see that

288
00:22:47,000 --> 00:22:51,000
they can't grow. All
right. This is what

289
00:22:51,000 --> 00:22:54,000
geneticists basically do. What
happens if I grew them on rich

290
00:22:54,000 --> 00:22:58,000
medium and I transferred them
to minimal medium? Why might

291
00:22:58,000 --> 00:23:02,000
something not grow? It might
be missing the ability to

292
00:23:02,000 --> 00:23:06,000
make tryptophan. It might
be missing the ability to

293
00:23:06,000 --> 00:23:10,000
make proline. It might be missing
the ability to make something else.

294
00:23:10,000 --> 00:23:14,000
So, what I can do is, if I wanted
to, make a very broad mutant hunt.

295
00:23:14,000 --> 00:23:19,000
I could just first grow on rich
medium and then plate on minimal

296
00:23:19,000 --> 00:23:23,000
medium and any yeast that has lost
the ability to make some essential

297
00:23:23,000 --> 00:23:27,000
nutrient will be evident by its
absence on the minimal medium plate.

298
00:23:27,000 --> 00:23:33,000
So, we have for yeasts.
Yeasts that are able to grow on

299
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minimal media are called prototrophs.
They are the wild type that can

300
00:23:39,000 --> 00:23:45,000
grow on minimal media. They
can make everything themselves.

301
00:23:45,000 --> 00:23:51,000
Yeasts that need help, that
cannot grow by themselves,

302
00:23:51,000 --> 00:23:57,000
that need help, that need a
supplement are called auxotrophs.

303
00:23:57,000 --> 00:24:03,000
Auxo obviously meaning help. So,
it's a mutant that has lost the

304
00:24:03,000 --> 00:24:09,000
ability to grow on minimal medium
and that it needs a supplement of

305
00:24:09,000 --> 00:24:15,000
some kind. So, if I wanted
to, I could just first

306
00:24:15,000 --> 00:24:21,000
collect lots and lots and lots of
auxotrophs and then figure out what

307
00:24:21,000 --> 00:24:27,000
they need. So, I might
collect a large collection

308
00:24:27,000 --> 00:24:33,000
of auxotrophs. And then
test to see if supplying

309
00:24:33,000 --> 00:24:40,000
arginine rescues them. I
could also test tryptophan.

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00:24:40,000 --> 00:24:48,000
So, if I only, only, only cared
about finding arginine auxotrophs,

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00:24:48,000 --> 00:24:55,000
I could just grow them on
minimal plus arginine and then

312
00:24:55,000 --> 00:25:00,000
test them on minimal. And
then I would know in advance,

313
00:25:00,000 --> 00:25:04,000
these guys all grew with arginine
on minimal and didn't grow without

314
00:25:04,000 --> 00:25:08,000
arginine, and I'd know
it was arginine. Or,

315
00:25:08,000 --> 00:25:11,000
if I was in an expansive mood,
I could test them on rich medium,

316
00:25:11,000 --> 00:25:15,000
collect everybody who's
unable to grow on minimal,

317
00:25:15,000 --> 00:25:19,000
and then work out what the
reason is. Is it arginine?

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00:25:19,000 --> 00:25:22,000
Is it proline? Is it whatever?
And it depends how much work you're

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00:25:22,000 --> 00:25:26,000
interested in doing and how
complete the study is you want to do.

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00:25:26,000 --> 00:25:30,000
Either way, we could end up with a
collection of arginine auxotrophs.

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00:25:30,000 --> 00:25:35,000
Organisms that are mutant for the
ability to make their own arginine

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00:25:35,000 --> 00:25:40,000
and require it to be supplied
to them in the medium.

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00:25:40,000 --> 00:25:45,000
All right. I might get,
depending on how much work I'm

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00:25:45,000 --> 00:25:50,000
willing to do, dozens
of independent colonies

325
00:25:50,000 --> 00:25:55,000
unable to grow without arginine.
I might get hundreds if I'm willing

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00:25:55,000 --> 00:26:00,000
to do enough work. I can
get as many as I want.

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00:26:00,000 --> 00:26:08,000
Our goal now is to study them and
find out why they're unable to do

328
00:26:08,000 --> 00:26:17,000
that. I have a quick question?
Those yeast cells we plated, where

329
00:26:17,000 --> 00:26:25,000
they haploid or diploid?
We didn't say, did we? So,

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00:26:25,000 --> 00:26:33,000
should they be haploid or
diploid? How many vote diploid?

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00:26:33,000 --> 00:26:39,000
How many vote haploid? A lot
of people vote haploid but

332
00:26:39,000 --> 00:26:45,000
aren't willing to express
a reason why. Why haploid?

333
00:26:45,000 --> 00:26:51,000
Right. Excellent. Excellent,
although genes are not

334
00:26:51,000 --> 00:26:57,000
recessive, but OK. A little
detail. Phenotypes are

335
00:26:57,000 --> 00:27:02,000
recessive. Tell me a
little more of what you're

336
00:27:02,000 --> 00:27:07,000
thinking about. We'll
have it out later on this

337
00:27:07,000 --> 00:27:12,000
point, yes. So, suppose we
were looking in a haploid.

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00:27:12,000 --> 00:27:17,000
I take your point, even if
on nomenclature I want to

339
00:27:17,000 --> 00:27:22,000
push back a bit. So,
suppose it's a diploid and

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00:27:22,000 --> 00:27:27,000
suppose we have now two
copies of this chromosome here

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00:27:27,000 --> 00:27:32,000
in the diploid. And suppose
there's a gene over here

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00:27:32,000 --> 00:27:36,000
that encodes an enzyme that we
now is necessary to make arginine,

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00:27:36,000 --> 00:27:41,000
or that somebody knows is
necessary to make arginine.

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00:27:41,000 --> 00:27:46,000
Let's image that that's the case.
In order to get haploid yeast that

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00:27:46,000 --> 00:27:50,000
is unable to make arginine
due to a mutation in this gene,

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00:27:50,000 --> 00:27:55,000
you need to have some kind
of a mutation in this copy.

347
00:27:55,000 --> 00:28:00,000
What about in the diploid yeast?
In order to make this yeast unable

348
00:28:00,000 --> 00:28:05,000
to grow without arginine, do we
need a mutation in both copies?

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00:28:05,000 --> 00:28:09,000
Well, the answer is probably.
The truth is actually a bit more

350
00:28:09,000 --> 00:28:13,000
complicated, but let's suppose it
was the case that even one copy of

351
00:28:13,000 --> 00:28:17,000
the functional gene was sufficient
to carry out the enzymatic step,

352
00:28:17,000 --> 00:28:21,000
then the answer would be yeah,
we'd need a mutation of both copies.

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00:28:21,000 --> 00:28:25,000
What's the chance of finding a
yeast that has a mutation in both

354
00:28:25,000 --> 00:28:29,000
copies? It's obviously much less
than the chance of finding a yeast

355
00:28:29,000 --> 00:28:33,000
that had a mutation of one
copy. So, we're much better to go

356
00:28:33,000 --> 00:28:37,000
searching in the haploid where the
phenotype will be revealed much more

357
00:28:37,000 --> 00:28:41,000
easily by virtue of just the single
mutation rather than having to,

358
00:28:41,000 --> 00:28:45,000
by chance, encounter one that
had mutations in both copies.

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00:28:45,000 --> 00:28:50,000
Now, the reason I'm a little
bit cautious here is because

360
00:28:50,000 --> 00:28:54,000
notwithstanding the textbooks,
it's not always the case that

361
00:28:54,000 --> 00:28:58,000
everything like this is a
recessive trait. It's possible that

362
00:28:58,000 --> 00:29:03,000
auxotrophy for arginine
could be a dominant trait.

363
00:29:03,000 --> 00:29:06,000
So, how could that be?
Well, auxotrophy could be a

364
00:29:06,000 --> 00:29:09,000
recessive trait. Suppose
there's some enzymatic

365
00:29:09,000 --> 00:29:12,000
pathway, A goes to B goes to C goes
to D, and this encodes an enzyme

366
00:29:12,000 --> 00:29:16,000
that carries out a
particular biochemical step.

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00:29:16,000 --> 00:29:19,000
Well, if the gene is broken, if
the gene is missing, if the gene

368
00:29:19,000 --> 00:29:22,000
doesn't make the protein, as
you guys all know that that's

369
00:29:22,000 --> 00:29:25,000
what happens, then you don't have
the enzyme, you can't do the pathway.

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00:29:25,000 --> 00:29:29,000
And it is usually the case that
having just one copy is sufficient.

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00:29:29,000 --> 00:29:32,000
Because having a little bit of
enzyme the pathway may work slower

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00:29:32,000 --> 00:29:35,000
but it will still work just fine and
you'll eventually get arginine made.

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00:29:35,000 --> 00:29:39,000
But it's occasionally possible,
I note since you guys are

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00:29:39,000 --> 00:29:42,000
sophisticated, that
sometimes a gene can encode a

375
00:29:42,000 --> 00:29:46,000
protein which not only doesn't
work but screws up the other working

376
00:29:46,000 --> 00:29:49,000
copies of the protein. Suppose
the enzyme that did this

377
00:29:49,000 --> 00:29:53,000
were a tetramer. It had
several subunits that had

378
00:29:53,000 --> 00:29:57,000
come together. A mutant
copy of an enzyme,

379
00:29:57,000 --> 00:30:03,000
when it forms into a tetramer,
might somehow disrupt all the other

380
00:30:03,000 --> 00:30:09,000
good copies that are around.
And that does happen sometimes.

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00:30:09,000 --> 00:30:14,000
It can happen that you're going to
have an inability to make your own

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00:30:14,000 --> 00:30:20,000
arginine be a dominantly inherited
trait. So, you actually have to

383
00:30:20,000 --> 00:30:26,000
test whether it's recessive
or dominant. Often it will be

384
00:30:26,000 --> 00:30:32,000
recessive. So, usually
most of these simple

385
00:30:32,000 --> 00:30:37,000
auxotrophs are recessive traits.
Occasionally some are dominant.

386
00:30:37,000 --> 00:30:43,000
So, now, suppose we get a whole
collection of Arg auxotrophs,

387
00:30:43,000 --> 00:30:49,000
and we'll just give them a name.
I don't know. Here's my collection.

388
00:30:49,000 --> 00:30:54,000
We'll call the first one,
for lack of anything terribly

389
00:30:54,000 --> 00:31:00,000
creative, Arg 1, Arg
2, Arg 3, et cetera,

390
00:31:00,000 --> 00:31:06,000
each being an individual strain from
growing up originally for a single

391
00:31:06,000 --> 00:31:12,000
colony that is unable to
produce its own arginine.

392
00:31:12,000 --> 00:31:17,000
We now want to take this
collection and characterize it.

393
00:31:17,000 --> 00:31:22,000
How many distinct genes does this
affect? Are these mutants perhaps

394
00:31:22,000 --> 00:31:28,000
all in the same gene? Are
they in a hundred different

395
00:31:28,000 --> 00:31:32,000
genes? How could we
tell? Now, of course,

396
00:31:32,000 --> 00:31:36,000
if you're a biochemist, you
already know the protein you can

397
00:31:36,000 --> 00:31:39,000
see and dah, dah, dah.
But, if you know the answer,

398
00:31:39,000 --> 00:31:42,000
well, why are asking then, right?
A geneticist goes out to ask this

399
00:31:42,000 --> 00:31:46,000
question because he or she wants to
know all the possible ways you can

400
00:31:46,000 --> 00:31:49,000
disrupt the cell so it cannot
make arginine. And we don't know in

401
00:31:49,000 --> 00:31:53,000
advance what those ways are, so
how are we going to be able to

402
00:31:53,000 --> 00:31:56,000
tell whether or not different
mutations affect the same gene,

403
00:31:56,000 --> 00:32:00,000
the same function in yeast?
It's an interesting question.

404
00:32:00,000 --> 00:32:10,000
Geneticists do a variety of tests.
The first test that a geneticist

405
00:32:10,000 --> 00:32:20,000
does to characterize a mutant is by
tests of recessivity or dominance,

406
00:32:20,000 --> 00:32:30,000
whichever way you
want to put it.

407
00:32:30,000 --> 00:32:33,000
We want to take each mutant and test
whether it is recessive or dominant

408
00:32:33,000 --> 00:32:37,000
as a phenotype,
whether the phenotype,

409
00:32:37,000 --> 00:32:40,000
the auxotrophy for arginine
is recessive or dominant.

410
00:32:40,000 --> 00:32:44,000
So, here's mutant number one,
the mutant cell carrying this

411
00:32:44,000 --> 00:32:47,000
mutation here. Conceptually
it affects some gene.

412
00:32:47,000 --> 00:32:51,000
I'm going to label it Arg 1. We
don't know where it is in the genome.

413
00:32:51,000 --> 00:32:54,000
There are other chromosomes here
as well. Here's my mutant cell.

414
00:32:54,000 --> 00:32:58,000
How am I going to find out whether
or not the auxotrophy for arginine

415
00:32:58,000 --> 00:33:03,000
is recessive or
dominant? Yup? With what?

416
00:33:03,000 --> 00:33:09,000
Cross it with a haploid that is
a prototroph, or I could just say

417
00:33:09,000 --> 00:33:15,000
cross it with wild type,
right? Perfect. So, make a cross

418
00:33:15,000 --> 00:33:21,000
here, very good, with
wild type plus there.

419
00:33:21,000 --> 00:33:27,000
How do I know it's plus there?
This is wild type. Wild type is

420
00:33:27,000 --> 00:33:32,000
defined as the normal form. And
so, because I said this is what

421
00:33:32,000 --> 00:33:37,000
we're using as wild type, it's
necessarily plus because we're

422
00:33:37,000 --> 00:33:42,000
measuring mutations relative to wild
type. So, what happens when we get

423
00:33:42,000 --> 00:33:47,000
here? We now, when we
cross we get a diploid,

424
00:33:47,000 --> 00:33:52,000
and Arg 1 plus. Now, how do we know
whether or not that phenotype was

425
00:33:52,000 --> 00:33:57,000
recessive or dominant? Sorry?
It's what shows up when we

426
00:33:57,000 --> 00:34:02,000
try to grow it. So,
when we cross it,

427
00:34:02,000 --> 00:34:07,000
what kind of plate should we grow
it on first? Should we grow it on

428
00:34:07,000 --> 00:34:12,000
minimal or rich? We better
grow it on rich because

429
00:34:12,000 --> 00:34:17,000
just in case it doesn't, it
can't make its own arginine,

430
00:34:17,000 --> 00:34:22,000
we better first let it grow and then
test it. So, let's grow it on rich

431
00:34:22,000 --> 00:34:27,000
medium. We'll cross these together,
grow it on rich medium. So, grow on

432
00:34:27,000 --> 00:34:32,000
rich, test on minimal. OK?
And we'll be able to check out

433
00:34:32,000 --> 00:34:36,000
the phenotype as to whether or not
the phenotype is wild type or mutant.

434
00:34:36,000 --> 00:34:41,000
All right. So,
we could do that.

435
00:34:41,000 --> 00:34:45,000
And we'll test the first one and
the second one and third one and the

436
00:34:45,000 --> 00:34:50,000
fourth one. And,
for each of these,

437
00:34:50,000 --> 00:34:55,000
we'll write down whether it's
recessive or a dominant auxotroph.

438
00:34:55,000 --> 00:34:59,000
Now, let me assume that all
the ones we're talking about are

439
00:34:59,000 --> 00:35:04,000
recessive phenotypes. Because
everything I'm about to say

440
00:35:04,000 --> 00:35:10,000
is very much harder if it turned
out any of them were dominant.

441
00:35:10,000 --> 00:35:16,000
So, we're going to assume.
Let's assume now, but it's not

442
00:35:16,000 --> 00:35:21,000
always the case, we'll
assume that the collection,

443
00:35:21,000 --> 00:35:27,000
maybe Arg 100, are all recessive
auxotrophies, the phenotype

444
00:35:27,000 --> 00:35:35,000
is recessive. Now, how do
I tell if they're in the

445
00:35:35,000 --> 00:35:45,000
same gene or not? So, now
I want to characterize my

446
00:35:45,000 --> 00:35:55,000
mutant by some other test that will
tell me whether or not Arg 1 and Arg

447
00:35:55,000 --> 00:36:03,000
2 are in the same gene.
Suppose Arg 1 and Arg 2 are in

448
00:36:03,000 --> 00:36:11,000
different genes. Cross
them. What will happen?

449
00:36:11,000 --> 00:36:19,000
Right. So, to repeat that, if I
cross together the two mutants and

450
00:36:19,000 --> 00:36:26,000
they're in different genes,
each will have at least, the each

451
00:36:26,000 --> 00:36:34,000
will be contributing a good copy,
a functional copy, a wild type copy

452
00:36:34,000 --> 00:36:40,000
of one of the genes. So,
let's walk this through.

453
00:36:40,000 --> 00:36:45,000
Interesting. Interesting. So,
suppose I take a situation where

454
00:36:45,000 --> 00:36:50,000
I've got Arg 1, a mutation
in a gene over here,

455
00:36:50,000 --> 00:36:55,000
on this chromosome, and on the other
chromosome I've got a wild type copy.

456
00:36:55,000 --> 00:37:00,000
My Arg 1 mutant is
mutated in a gene here.

457
00:37:00,000 --> 00:37:07,000
I've got this other gene here,
which is normal. And I'm going to

458
00:37:07,000 --> 00:37:14,000
cross that now by the strain that
has a wild type copy here for this

459
00:37:14,000 --> 00:37:21,000
first gene, but it has a
mutation in the second gene.

460
00:37:21,000 --> 00:37:28,000
When I cross them together, I
now get me a diploid cell here,

461
00:37:28,000 --> 00:37:35,000
which is Arg 1, a mutation
there, plus there, plus copy

462
00:37:35,000 --> 00:37:41,000
here, and Arg 2.
Will having one copy,

463
00:37:41,000 --> 00:37:47,000
one working copy of this gene
be enough to make the enzyme?

464
00:37:47,000 --> 00:37:53,000
No? In other words, is the wild
type phenotype dominant to this

465
00:37:53,000 --> 00:37:59,000
auxotrophy, or is the
auxotrophy attributable to this

466
00:37:59,000 --> 00:38:04,000
gene recessive? Yes. Why?
Because we assumed it.

467
00:38:04,000 --> 00:38:09,000
Why did we assume it? So I
would be able to say this,

468
00:38:09,000 --> 00:38:14,000
right? OK. If it wasn't we'd be in
trouble. But by assuming that we're

469
00:38:14,000 --> 00:38:19,000
working with a recessive phenotype,
then we know that this will be

470
00:38:19,000 --> 00:38:24,000
enough to save the yeast.
What about here? Enough to save

471
00:38:24,000 --> 00:38:30,000
the yeast so it will
grow without arginine.

472
00:38:30,000 --> 00:38:38,000
By contrast, suppose it was
the case that this cell here,

473
00:38:38,000 --> 00:38:47,000
Arg 1, and suppose our other mutant
that we had isolated in our mutant

474
00:38:47,000 --> 00:38:56,000
hunt was a mutation Arg 2 in the
same gene. Suppose these were the

475
00:38:56,000 --> 00:39:05,000
same gene. When I cross them
together I now have a cell

476
00:39:05,000 --> 00:39:14,000
that is Arg 1, Arg
2. In other words,

477
00:39:14,000 --> 00:39:24,000
its genotype is Arg 1 over Arg 2,
name of mutation. And can it grow?

478
00:39:24,000 --> 00:39:34,000
No growth without arginine. By
contrast, the genotype here is Arg 1

479
00:39:34,000 --> 00:39:43,000
over plus, plus over Arg 2. I
could even write Arg 2 over plus,

480
00:39:43,000 --> 00:39:51,000
but I just did that to indicate
the chromosomes that they came from.

481
00:39:51,000 --> 00:39:59,000
All right. This is called a Test
of Complementation because these two

482
00:39:59,000 --> 00:40:07,000
genes are able to compliment
each other's defect.

483
00:40:07,000 --> 00:40:18,000
If two mutations compliment each
other's defect then they are in

484
00:40:18,000 --> 00:40:30,000
different genes. OK?
Boy, that's a noisy one.

485
00:40:30,000 --> 00:40:36,000
So, we're able to make
a Complementation Table.

486
00:40:36,000 --> 00:40:43,000
Suppose I take a bunch of yeasts,
wild type, WT, mutant number one,

487
00:40:43,000 --> 00:40:49,000
mutant number two,
mutant number three,

488
00:40:49,000 --> 00:40:56,000
mutant number four. And suppose I
cross them with each other in all

489
00:40:56,000 --> 00:41:02,000
pair-wise combinations. I've
assumed that all of these

490
00:41:02,000 --> 00:41:07,000
arginine auxotrophs have
a recessive phenotype here.

491
00:41:07,000 --> 00:41:12,000
These are all my Arg mutants,
and I'm assuming that this is

492
00:41:12,000 --> 00:41:17,000
recessive. What happens when I
cross them and I test to see whether

493
00:41:17,000 --> 00:41:22,000
they can grow without arginine?
If I cross wild type by wild type,

494
00:41:22,000 --> 00:41:27,000
can it grow without arginine?
Yeah. Normal phenotype. So, plus is

495
00:41:27,000 --> 00:41:32,000
going to mean prototrophic.
Minus will mean auxotrophic for

496
00:41:32,000 --> 00:41:36,000
arginine. What happens when I cross
wild type with mutant number one?

497
00:41:36,000 --> 00:41:41,000
It grows. Why? By assumption,
these were all recessive.

498
00:41:41,000 --> 00:41:46,000
I'm only testing recessive ones.
Two. Three. Four. When I cross

499
00:41:46,000 --> 00:41:50,000
in this direction,
wild type by these guys.

500
00:41:50,000 --> 00:41:55,000
This is going to be a
symmetric matrix, of course,

501
00:41:55,000 --> 00:42:00,000
right? OK. Now, what happens when
I cross mutant one by mutant one?

502
00:42:00,000 --> 00:42:04,000
I now have a diploid. Will
it be able to grow without

503
00:42:04,000 --> 00:42:08,000
arginine? No. Why not?
It has no working copies

504
00:42:08,000 --> 00:42:12,000
of that gene, so I'm going to put
a minus there. What about mutant two

505
00:42:12,000 --> 00:42:16,000
with mutant two? Minus.
What about mutant three

506
00:42:16,000 --> 00:42:20,000
with mutant three? Minus.
What about mutant four with

507
00:42:20,000 --> 00:42:24,000
mutant four? Minus. Now,
what happens when I cross

508
00:42:24,000 --> 00:42:28,000
mutant one by mutant two? It
depends. It might be plus or

509
00:42:28,000 --> 00:42:32,000
might be minus. If
they're in the same gene,

510
00:42:32,000 --> 00:42:38,000
minus. Different genes, could
be plus. So, here's some data.

511
00:42:38,000 --> 00:42:43,000
So, all this is compelled.
But the kind of data, ooh,

512
00:42:43,000 --> 00:42:49,000
I'll use a color. Isn't that fun?
They want me to use colors over

513
00:42:49,000 --> 00:42:54,000
there. Here we go. Suppose
the data were minus,

514
00:42:54,000 --> 00:43:00,000
minus, plus, plus, plus, plus,
minus, minus, plus, plus, plus,

515
00:43:00,000 --> 00:43:06,000
plus. What
would it be?

516
00:43:06,000 --> 00:43:13,000
What conclusion could we draw?
Is mutant one and mutant three in

517
00:43:13,000 --> 00:43:20,000
the same gene? They
compliment each other?

518
00:43:20,000 --> 00:43:27,000
No. But is one in the same
gene as two? Yes. In fact,

519
00:43:27,000 --> 00:43:34,000
this box and this box here
define the genes beautifully.

520
00:43:34,000 --> 00:43:37,000
The groups that failed to compliment
define mutations in the same gene.

521
00:43:37,000 --> 00:43:41,000
These are called Complementation
Groups because they don't compliment,

522
00:43:41,000 --> 00:43:45,000
OK? It's a little complicated
but that's all right.

523
00:43:45,000 --> 00:43:48,000
These are called Complementation
Groups because all the members of

524
00:43:48,000 --> 00:43:52,000
the complementation group,
namely Arg 1 and Arg 2, failed to

525
00:43:52,000 --> 00:43:56,000
compliment each other. They
could be called failure to

526
00:43:56,000 --> 00:44:00,000
compliment groups, but
it would be too long.

527
00:44:00,000 --> 00:44:04,000
OK? So, there you go. You
can take hundreds of mutants

528
00:44:04,000 --> 00:44:09,000
and organize them into
complementation groups and thereby

529
00:44:09,000 --> 00:44:13,000
know which ones go to the same
gene. And now, if I want to study the

530
00:44:13,000 --> 00:44:18,000
genes, I only have to study the
distinct complementation groups.

531
00:44:18,000 --> 00:44:23,000
Last thing, which we'll just have
time to do, are what's called tests

532
00:44:23,000 --> 00:44:28,000
of epistasis. We'll probably run
just a moment or two over on this.

533
00:44:28,000 --> 00:44:34,000
Suppose a biochemist were
collaborating with a geneticist and

534
00:44:34,000 --> 00:44:40,000
had studied what he or she thought
was the pathway for making arginine.

535
00:44:40,000 --> 00:44:47,000
Some precursor alpha
goes to precursor beta,

536
00:44:47,000 --> 00:44:53,000
goes to precursor gamma,
goes to arginine. And suppose

537
00:44:53,000 --> 00:45:00,000
specific genes were needed
to encode specific proteins.

538
00:45:00,000 --> 00:45:05,000
I'll call them Arg A, Arg B,
Arg C to catalyze each step

539
00:45:05,000 --> 00:45:10,000
of this biochemical reaction.
The geneticist and the biochemist

540
00:45:10,000 --> 00:45:15,000
could collaborate with each other
to study whether these mutants,

541
00:45:15,000 --> 00:45:20,000
these particular genes now
that had been identified,

542
00:45:20,000 --> 00:45:26,000
affected each step of the pathway.
And here's how they might do it.

543
00:45:26,000 --> 00:45:30,000
They might take wild type yeast,
mutant, well, they wouldn't know in

544
00:45:30,000 --> 00:45:34,000
advance whether or not it was
missing the ability to grow on each

545
00:45:34,000 --> 00:45:38,000
of, whether it was missing
each of these enzymes,

546
00:45:38,000 --> 00:45:42,000
but let's think conceptually.
Suppose we had a mutant that was,

547
00:45:42,000 --> 00:45:46,000
a strain that was wild type,
Arg A minus, Arg B, minus,

548
00:45:46,000 --> 00:45:50,000
Arg C minus, unable to make
this enzyme, this enzyme,

549
00:45:50,000 --> 00:45:54,000
this enzyme. And suppose we helped
it along. Suppose we gave the

550
00:45:54,000 --> 00:45:58,000
mutant arginine. Suppose
we supplement and grow it

551
00:45:58,000 --> 00:46:02,000
on media with arginine. Which
ones will be able to grow with

552
00:46:02,000 --> 00:46:08,000
arginine? Can wild type
grow if it's given arginine?

553
00:46:08,000 --> 00:46:13,000
What about Arg A minus? B
minus? C minus? What if instead

554
00:46:13,000 --> 00:46:19,000
we offer it precursor gamma?
Will wild type be able to grow if

555
00:46:19,000 --> 00:46:24,000
it's given precursor gamma?
Sure. What about Arg A minus?

556
00:46:24,000 --> 00:46:30,000
No, because it still
is stuck at this step.

557
00:46:30,000 --> 00:46:35,000
It cannot. What about Arg B minus?
What about Arg C minus? Really?

558
00:46:35,000 --> 00:46:41,000
It hasn't got this enzyme.
What's it going to do with gamma?

559
00:46:41,000 --> 00:46:47,000
It ain't got anything to
do with gamma, no enzyme.

560
00:46:47,000 --> 00:46:53,000
Suppose I gave it beta. Wild
type, can it grow? What about

561
00:46:53,000 --> 00:46:59,000
Arg A minus? No, because it
can go from alpha to beta,

562
00:46:59,000 --> 00:47:04,000
but it can't go to
gamma. It cannot grow.

563
00:47:04,000 --> 00:47:10,000
What about Arg B minus? I've
given it beta, but it can't do

564
00:47:10,000 --> 00:47:16,000
anything with beta because
it hasn't got this gene.

565
00:47:16,000 --> 00:47:21,000
What about Arg C minus? Wait
a second. What did I just do?

566
00:47:21,000 --> 00:47:27,000
We're just backward. Sorry.
If we gave it gamma, I just

567
00:47:27,000 --> 00:47:32,000
got lost here. If we gave
it gamma it was able to

568
00:47:32,000 --> 00:47:36,000
grow, well, we are completely
wrong, guys. It's able to grow here.

569
00:47:36,000 --> 00:47:40,000
Thank you. Let's go back on that.
You should have caught me before.

570
00:47:40,000 --> 00:47:44,000
My mistake. If we have it gamma
it's able to, if it's a mutant here

571
00:47:44,000 --> 00:47:48,000
it can grow because it bypasses this
problem. And having gamma is enough.

572
00:47:48,000 --> 00:47:52,000
If I gave it beta, sorry,
if I gave it gamma and its

573
00:47:52,000 --> 00:47:57,000
mutation was here
it can grow. Sorry.

574
00:47:57,000 --> 00:48:02,000
Now, if I gave it here beta,
and its mutation was here, it can

575
00:48:02,000 --> 00:48:07,000
still grow, right? But
if its mutation is here it

576
00:48:07,000 --> 00:48:12,000
can't and if its mutation is
here it can't. That's better.

577
00:48:12,000 --> 00:48:17,000
I was getting worried there for
a while myself. Suppose I gave it

578
00:48:17,000 --> 00:48:22,000
alpha. Wild type can grow. If
I give this guy alpha, will that

579
00:48:22,000 --> 00:48:27,000
help if he's mutant in A? No.
Can it help if he's mutant in

580
00:48:27,000 --> 00:48:32,000
B? No. Can it help
if he's mutant in C?

581
00:48:32,000 --> 00:48:37,000
No. Sorry. There we go. I
usually start at the other end of

582
00:48:37,000 --> 00:48:42,000
this picture. So, what you
can see is these mutants

583
00:48:42,000 --> 00:48:47,000
have different phenotypes with
respect to being able to supplement

584
00:48:47,000 --> 00:48:52,000
them with different chemicals.
Now, let me ask in our last two

585
00:48:52,000 --> 00:48:57,000
minutes, I'll run two minutes over
here. Suppose I gave you a mutant

586
00:48:57,000 --> 00:49:03,000
that was a double homozygote.
Suppose it was Arg B minus,

587
00:49:03,000 --> 00:49:09,000
Arg B minus, sorry, Arg B minus
and Arg C minus. Suppose it was a

588
00:49:09,000 --> 00:49:16,000
double mutant, it lacked
both this and this.

589
00:49:16,000 --> 00:49:23,000
Which line of my table would it
resemble? Would it look like the

590
00:49:23,000 --> 00:49:30,000
first line, the second line
or the third line of my table?

591
00:49:30,000 --> 00:49:36,000
Second line. Why's that? If
I'm lacking B, I'm already in

592
00:49:36,000 --> 00:49:42,000
trouble here. And also
lacking C doesn't matter.

593
00:49:42,000 --> 00:49:49,000
So, I will look, just
like a mutant who lacks B.

594
00:49:49,000 --> 00:49:55,000
So, in other words, I'm able,
if I know something about the

595
00:49:55,000 --> 00:50:02,000
biochemistry of a pathway and I can
break my arginine mutants up into

596
00:50:02,000 --> 00:50:08,000
different kinds of phenotypes here
by their response to different steps

597
00:50:08,000 --> 00:50:15,000
in a pathway, I can then look
at combinations of mutants.

598
00:50:15,000 --> 00:50:20,000
And I can say if I have a double
mutant missing both B and C,

599
00:50:20,000 --> 00:50:25,000
does it look like B or does it
look C when I put them together?

600
00:50:25,000 --> 00:50:31,000
And it turns out that if it looks
like B then B was further upstream

601
00:50:31,000 --> 00:50:36,000
in the pathway. So, it
turns out that geneticists

602
00:50:36,000 --> 00:50:40,000
and biochemists can collaborate
based on the phenotype of the

603
00:50:40,000 --> 00:50:45,000
organism sometimes to infer
aspects of the biochemical pathway.

604
00:50:45,000 --> 00:50:49,000
These are the kinds of things
a geneticist does to be able to

605
00:50:49,000 --> 00:50:54,000
characterize mutants on a mutant
hunt. Next time what I want to do

606
00:50:54,000 --> 00:50:58,000
is talk about characterizing mutants
in a very different kind of organism,

607
00:50:58,000 --> 00:51:03,000
namely the
human being.