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00:00:00,100 --> 00:00:05,000
So I just want to back up a couple
because I think there's still some

2
00:00:05,100 --> 00:00:11,000
confusion about what a restriction
enzyme is and exactly what it does,

3
00:00:11,100 --> 00:00:16,000
although I indicated, a three prime
hydroxyl and a five-prime phosphate.

4
00:00:16,100 --> 00:00:22,000
Let me show you.  This is the way a
lot, at least a large class of

5
00:00:22,100 --> 00:00:27,000
restriction enzymes,
went.  We've seen a deoxyribose

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00:00:27,100 --> 00:00:33,000
backbone before with a phosphate
backbone going on up to

7
00:00:33,100 --> 00:00:40,000
the next nucleotide.
This is the five-prime position.

8
00:00:40,100 --> 00:00:48,000
This is the three prime position.
There is a phosphate here.  It goes

9
00:00:48,100 --> 00:00:57,000
down to the next one.
It goes down like this.

10
00:00:57,100 --> 00:01:06,000
So what I'm showing you,
GAATTC, five prime to three prime,

11
00:01:06,100 --> 00:01:15,000
here's where I indicated the first
cut comes from.

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00:01:15,100 --> 00:01:20,000
And I indicated that the cleavage
generates a G with a three prime

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00:01:20,100 --> 00:01:26,000
hydroxyl, and the G ends up with a
five prime phosphate.

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00:01:26,100 --> 00:01:32,000
So what that means is that the
hydrolysis happens right there,

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00:01:32,100 --> 00:01:38,000
so that after cleavage, what you end
up with is a five-prime phosphate

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00:01:38,100 --> 00:01:44,000
and three prime hydroxyl.
This is paired with the C on the

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00:01:44,100 --> 00:01:51,000
opposite strand.
It makes one cut here,

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00:01:51,100 --> 00:01:57,000
and then it would make an identical
cut on the opposite strand.

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00:01:57,100 --> 00:02:03,000
So if we were to pull those apart
this strand here would have G with

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00:02:03,100 --> 00:02:10,000
its three prime OH,
and the other strand would be TTAA.

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00:02:10,100 --> 00:02:16,000
And if we were to pull the apart.
We'd have A A T T C, a three prime

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00:02:16,100 --> 00:02:23,000
OH here, five prime phosphate there,
five prime phosphate there.  Again,

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00:02:23,100 --> 00:02:29,000
you have to remember,
this strand is going five prime,

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00:02:29,100 --> 00:02:36,000
three prime in that direction.  This
was going five prime, three

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00:02:36,100 --> 00:02:42,000
prime in the end.
And the beauty of these restriction

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00:02:42,100 --> 00:02:46,000
enzymes, at least it's not true of
all of them.  But it's true of a lot

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00:02:46,100 --> 00:02:50,000
of them as they generate these what
are called sticky ends.

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00:02:50,100 --> 00:02:54,000
You can pull them apart.
They come back together, though,

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00:02:54,100 --> 00:02:58,000
and reform those base pairs.  It's
almost like having little bits of

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00:02:58,100 --> 00:03:02,000
Velcro at the end.
And when this end is looking for a

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00:03:02,100 --> 00:03:06,000
complementary sequence to pair with.
It doesn't know what's out here,

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00:03:06,100 --> 00:03:10,000
and it doesn't know what's out there,
all it sees as this.

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00:03:10,100 --> 00:03:14,000
So I can cut this and rejoin it or
I can cut them and pull apart,

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00:03:14,100 --> 00:03:19,000
take another piece of DNA that's
been cut with the same enzyme and

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00:03:19,100 --> 00:03:23,000
therefore has the same corresponding
little sticky ends on each end,

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00:03:23,100 --> 00:03:27,000
and it could insert right in the
middle.  And that's the

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00:03:27,100 --> 00:03:32,000
principle of cloning.
And it was the development of these,

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00:03:32,100 --> 00:03:38,000
if you will, magic scissors that
made it possible to take this DNA

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00:03:38,100 --> 00:03:43,000
which looks so homogeneous,
nothing but GA's, T's, and C's,

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00:03:43,100 --> 00:03:49,000
and then cut it up in defined ways.
When I was a postdoc, a friend of

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00:03:49,100 --> 00:03:54,000
mine had just purified $2 million
worth of EcoRI because he purified

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00:03:54,100 --> 00:04:00,000
some of this enzyme.
At that point,

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00:04:00,100 --> 00:04:04,000
the only way to get these things was
to produce them yourself and purify

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00:04:04,100 --> 00:04:08,000
them.  Now there are literally
hundreds of these and they recognize

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00:04:08,100 --> 00:04:12,000
different sequences,
and once people understood that they

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00:04:12,100 --> 00:04:16,000
existed, then they just started to
look in different,

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00:04:16,100 --> 00:04:20,000
usually they're from bacteria,
and they just looked in different

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00:04:20,100 --> 00:04:24,000
bacteria until they found another
one.  And then they purified it.

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00:04:24,100 --> 00:04:28,000
So if you go to, say, any of the
companies that do stuff for

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00:04:28,100 --> 00:04:32,000
recombinant DNA, you'll
find lists like this.

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00:04:32,100 --> 00:04:36,000
Funny little abbreviations like
EcoRI usually have something that

52
00:04:36,100 --> 00:04:40,000
tells you some abbreviation related
to the organism,

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00:04:40,100 --> 00:04:45,000
from which the restriction enzyme
was isolated.  And you can find

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00:04:45,100 --> 00:04:49,000
things that will cut,
I won't say every sequence,

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00:04:49,100 --> 00:04:54,000
but very, very, many sequences.
There are literally hundreds of

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00:04:54,100 --> 00:04:58,000
these, and you just order them.
And the next day, a FedEx package

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00:04:58,100 --> 00:05:02,000
arrives with a little bit of the
enzyme that will cut

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00:05:02,100 --> 00:05:08,000
at that sequence.
Another concept that seemed to be a

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00:05:08,100 --> 00:05:16,000
problem, was what's a vector?
So if you understand that there are

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00:05:16,100 --> 00:05:23,000
sequence specific molecular scissors,
that if we have a piece of DNA and

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00:05:23,100 --> 00:05:31,000
there's an EcoRI site here,
you would come to think of it like

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00:05:31,100 --> 00:05:39,000
that because it's going to cut in a
slightly skewed way.

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00:05:39,100 --> 00:05:44,000
Maybe there's another one right here,
another one over here.

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00:05:44,100 --> 00:05:50,000
If we take this DNA and cut it with
this particular enzyme,

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00:05:50,100 --> 00:05:56,000
we get a break here.  Then this will
get this piece running from here to

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00:05:56,100 --> 00:06:02,000
here.  We'll get this little piece
here.  We'll get this piece here,

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00:06:02,100 --> 00:06:08,000
and will get whatever goes off on
those sides.  So that's naked

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00:06:08,100 --> 00:06:13,000
DNA in a test tube.
So I could cut any piece of DNA at

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00:06:13,100 --> 00:06:19,000
some sites generating a bunch of
fragments and if I just took those

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00:06:19,100 --> 00:06:24,000
fragments and transformed them into
E. coli, I took naked DNA,

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00:06:24,100 --> 00:06:30,000
took it from the outside, put it
inside, is it going

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00:06:30,100 --> 00:06:35,000
to replicate?  No.
Why not?  Because there's a special

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00:06:35,100 --> 00:06:39,000
signal called the origin of
replication that says "start

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00:06:39,100 --> 00:06:43,000
replicating DNA here".
This came from a piece of human,

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00:06:43,100 --> 00:06:47,000
lets say some of my DNA, it would
not have a signal in it that said to

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00:06:47,100 --> 00:06:52,000
the E. coli replication machinery,
"start replicating DNA right here".

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00:06:52,100 --> 00:06:56,000
So the principle of, apart from
being able to cut DNA fragments,

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00:06:56,100 --> 00:07:00,000
is you have together to replicate so
you can make lots of

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00:07:00,100 --> 00:07:05,000
lots of copies.
The trick is to attach the DNA,

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00:07:05,100 --> 00:07:11,000
at least a widely used trick is to
attach the DNA to something that has

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00:07:11,100 --> 00:07:16,000
an origin of replication that will
work in the organism in question.

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00:07:16,100 --> 00:07:21,000
And that was what we call a vector.
So this is an E. coli cell.

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00:07:21,100 --> 00:07:27,000
Another thing that's very confusing
is all the circles that show up in

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00:07:27,100 --> 00:07:32,000
this course.  This is huge.
The vector was double-stranded DNA

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00:07:32,100 --> 00:07:36,000
that maybe, let's say,
had a unique EcoRI restriction site

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00:07:36,100 --> 00:07:40,000
in it.  That's the only EcoRI
restriction site.

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00:07:40,100 --> 00:07:44,000
The other thing that we'd need to
have is an origin of DNA replication.

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00:07:44,100 --> 00:07:48,000
So, that's why this plasmid is able
to propagate itself.

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00:07:48,100 --> 00:07:52,000
This little circle of DNA is able
to propagate itself,

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00:07:52,100 --> 00:07:56,000
and then some kind of selectable
marker.  And, most of the time

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00:07:56,100 --> 00:08:00,000
that's a drug-resistance,
so although it doesn't have to be.

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00:08:00,100 --> 00:08:05,000
And if we cut that here,

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00:08:05,100 --> 00:08:13,000
generate sticky ends, then we can
take this fragment and stick it in

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00:08:13,100 --> 00:08:20,000
here, to give this piece here joined
to the vector.

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00:08:20,100 --> 00:08:28,000
And that's an insert.
Let's say it was that piece there.

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00:08:28,100 --> 00:08:31,000
In fact, if you wanted to clone DNA
in E. coli, and then have the

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00:08:31,100 --> 00:08:35,000
plasmid work in yeast,
if you just take that plasmid that

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00:08:35,100 --> 00:08:38,000
works, the vector with its insert
that works in E.

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00:08:38,100 --> 00:08:42,000
coli and put it in the yeast,
it won't replicate either.  And

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00:08:42,100 --> 00:08:45,000
that's because these other languages
other than the genetic code are not

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00:08:45,100 --> 00:08:49,000
universal.  So,
you also have to put also in a

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00:08:49,100 --> 00:08:52,000
sequence that said to the yeast
replication machinery "start

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00:08:52,100 --> 00:08:56,000
something here".
People call that a shuttle vector,

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00:08:56,100 --> 00:08:59,000
something that will replicate in E.
coli or replicate in yeast,

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00:08:59,100 --> 00:09:03,000
and the same principle applies to
other organisms

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00:09:03,100 --> 00:09:07,000
Okay, now probably the trickiest
thing, the thing where I sort of

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00:09:07,100 --> 00:09:11,000
muddled it on Friday,
and I apologize for that,

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00:09:11,100 --> 00:09:15,000
was this discovery of restriction
enzymes.  And again,

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00:09:15,100 --> 00:09:19,000
some of you were frustrated.
You said, why do I waste time?

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00:09:19,100 --> 00:09:23,000
Why not just tell you stuff that's
on the exam?  Okay again,

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00:09:23,100 --> 00:09:27,000
people were talking about molecular
scissors when I was an undergrad and

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00:09:27,100 --> 00:09:31,000
grad student, and chemists were
trying to think if they could come

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00:09:31,100 --> 00:09:36,000
up with some way to get some
specificity in how to cutting DNA.

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00:09:36,100 --> 00:09:40,000
And the answer,
the discovery of restriction enzymes

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00:09:40,100 --> 00:09:44,000
didn't come from that kind of
experiment.  It came from somebody

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00:09:44,100 --> 00:09:48,000
trying to understand what seemed to
be a really obscure piece of biology.

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00:09:48,100 --> 00:09:52,000
Julie made up a lovely little slide.
But I think it basically did was I

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00:09:52,100 --> 00:09:56,000
left at one of the little layers
that I usually show.

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00:09:56,100 --> 00:10:03,000
So, let me just talk.
Here's, again, what Luria saw when

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00:10:03,100 --> 00:10:10,000
he was doing these experiments.
So, I'm going to tell you now what

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00:10:10,100 --> 00:10:17,000
was in strain A and B,
and maybe that will help.

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00:10:17,100 --> 00:10:24,000
But I wanted you first to see it
without knowing what anything beyond

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what Luria knew.
OK, strain A has no restriction

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enzyme, and no modification
enzyme.

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00:10:38,100 --> 00:10:48,000
And although there are different
types of modification enzymes,

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00:10:48,100 --> 00:10:58,000
many of them are methylases.  So,
we'll call that.  And this one has a

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00:10:58,100 --> 00:11:06,000
restriction enzyme.
And, it has corresponding methylase.

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00:11:06,100 --> 00:11:12,000
Just to review that again, if you
can figure this pretty much out from

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00:11:12,100 --> 00:11:18,000
first principles that if you were an
organism, and you had a restriction

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00:11:18,100 --> 00:11:24,000
enzyme that would cut this,
there are two things you can

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possibly do to keep from cutting up
your own DNA.

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00:11:31,100 --> 00:11:35,000
One is to never have that sequence
of pure DNA.  That would prevent you

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00:11:35,100 --> 00:11:39,000
from cutting up your own DNA,
even though it has it.  It's pretty

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00:11:39,100 --> 00:11:43,000
constraining now,
because somewhere in a particular

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00:11:43,100 --> 00:11:48,000
protein, you might need that little
bit of sequence to encode something

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00:11:48,100 --> 00:11:52,000
that you need to make a critical
protein.  So instead what you find

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00:11:52,100 --> 00:11:56,000
is organisms have a restriction
enzyme, have those sequences,

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00:11:56,100 --> 00:12:00,000
but they don't cut up their own DNA
because they modify their own DNA,

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00:12:00,100 --> 00:12:05,000
by putting, in the case of this one,
they put a methyl here.

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00:12:05,100 --> 00:12:09,000
And I drew that out the other day.
You can see, you can put a methyl

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00:12:09,100 --> 00:12:13,000
group on the exocyclic amino group
of adanine, and not interfere with

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00:12:13,100 --> 00:12:17,000
base pairing.  But what you can do,
is interfere with the way that the

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00:12:17,100 --> 00:12:21,000
restriction enzymes sees that
sequence.  And when the cell pulls

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00:12:21,100 --> 00:12:25,000
the DNA apart,
each of the old strands is

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00:12:25,100 --> 00:12:29,000
methylated, and a new strand is
initially not methylated.

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00:12:29,100 --> 00:12:33,000
But that's enough keep the
restriction enzyme from

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00:12:33,100 --> 00:12:37,000
doing its thing.
And then the methylase will come

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00:12:37,100 --> 00:12:41,000
along, find a sequence,
and then the progeny strand,

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00:12:41,100 --> 00:12:44,000
the daughter strand, will begin to
get methylated.

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00:12:44,100 --> 00:12:48,000
So, once you get DNA methylated,
you can propagate it as long as you

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00:12:48,100 --> 00:12:51,000
have the methyl group.
So this was what was really

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underlying what Luria did.
But he didn't know that.

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Let me quickly just go through this
again.  So he grew the strain,

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00:13:00,100 --> 00:13:05,000
the phage on strain A, no
restriction enzyme,

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00:13:05,100 --> 00:13:10,000
just plain old DNA.  So,
he picks a plaque that's probably

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00:13:10,100 --> 00:13:15,000
about a billion phage in a plaque,
somewhere around 10^8, 10^9 probably,

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no that's not true,
a little less than that,

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somewhat less than that, but lots
and lots of phage particles in the

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plaque. Resuspend them and
then plate them out.

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And of course it grew on strain A.
That's what it was growing on.

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00:13:34,100 --> 00:13:38,000
That wouldn't surprise you at all.
The surprise was, even though he

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00:13:38,100 --> 00:13:42,000
knew he had lots and lots of phage,
hardly any of them grew on strain B.

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00:13:42,100 --> 00:13:47,000
But, he found a rare plaque that
had learned to grow.

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00:13:47,100 --> 00:13:51,000
And he tested then, and this thing
grows on strain B.

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00:13:51,100 --> 00:13:55,000
That was not a surprise because it
had to grow on strain B to be up

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there.  And he tested,
and it still grew on the strain A.

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00:14:00,100 --> 00:14:04,000
So up until now,
everything we knew,

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00:14:04,100 --> 00:14:09,000
everything we've talked about in
that course, you think,

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00:14:09,100 --> 00:14:13,000
ah-ha, this original phage couldn't
grow on strain B,

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00:14:13,100 --> 00:14:18,000
but it's mutated. Somehow.
It's learned to grow on strain B.

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00:14:18,100 --> 00:14:22,000
That would be a perfectly
reasonable explanation.

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00:14:22,100 --> 00:14:27,000
But if that were the case,
what would've happened?

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00:14:27,100 --> 00:14:31,000
Well it was growing on those phage,
you pick them again, it should still

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00:14:31,100 --> 00:14:35,000
grow on A and B,
and they still do.

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00:14:35,100 --> 00:14:39,000
The problem with that model was
that they grew on strain A.

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00:14:39,100 --> 00:14:43,000
They had trouble growing on strain
B.  Now that we've learned to grow

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00:14:43,100 --> 00:14:47,000
on strain B, but you hadn't
forgotten how to grow on strain A.

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00:14:47,100 --> 00:14:51,000
But if you take the ones that were
growing on strain A,

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00:14:51,100 --> 00:14:56,000
they grow on strain A,
not a surprise.

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00:14:56,100 --> 00:15:00,000
This is the problem right here.
If this was a mutant, permanent

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00:15:00,100 --> 00:15:04,000
change, then we should have been
able to have it grow on strain B as

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00:15:04,100 --> 00:15:08,000
well.  So what was really happening
in there?  Well,

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00:15:08,100 --> 00:15:12,000
at the beginning,
the phage DNA was lacking any kind

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00:15:12,100 --> 00:15:16,000
of modification.
It grew fine on strain A,

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00:15:16,100 --> 00:15:20,000
because there was no restriction
enzyme.  When it went into strain B,

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00:15:20,100 --> 00:15:24,000
that now had an enzyme, a
restriction enzyme that cut up any

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00:15:24,100 --> 00:15:29,000
time it found that sequence.
And so, most of the phage that

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00:15:29,100 --> 00:15:33,000
injected their DNA,
those DNAs were trashed by the

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00:15:33,100 --> 00:15:37,000
restriction enzyme.
But there is a methylase in there

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00:15:37,100 --> 00:15:41,000
that's also able to methylate those
sequences.  And what happened

191
00:15:41,100 --> 00:15:45,000
somewhere along the way was that
there was the methyl,

192
00:15:45,100 --> 00:15:50,000
on one phage DNA, and got enough
methyls on there that the phage

193
00:15:50,100 --> 00:15:54,000
could be replicated before it got
cut up by the restriction enzyme.

194
00:15:54,100 --> 00:15:58,000
Once the phage molecule has
methylations on the site,

195
00:15:58,100 --> 00:16:02,000
it's able to grow just fine on
strain B, and it will be able to do

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00:16:02,100 --> 00:16:07,000
that forever.
However, if you take that DNA with

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00:16:07,100 --> 00:16:11,000
the methyls on it,
and we put it back strain A,

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00:16:11,100 --> 00:16:15,000
it'll still grow, as this one
doesn't have any kind of restriction

199
00:16:15,100 --> 00:16:19,000
enzyme, but while it's growing its
busy losing all the methyls again.

200
00:16:19,100 --> 00:16:23,000
We are right back to where we
started from.  An obscure experiment,

201
00:16:23,100 --> 00:16:27,000
one of the most obscure you could
get, many people would have paid no

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00:16:27,100 --> 00:16:31,000
attention.  It does
not seem worth it.

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00:16:31,100 --> 00:16:35,000
So, the phenomenon was called
restriction.  They had to give it a

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00:16:35,100 --> 00:16:39,000
name.  It wasn't mutation.
They said this phage DNA was being

205
00:16:39,100 --> 00:16:44,000
restricted somehow when it grew on
strain B.  People called them,

206
00:16:44,100 --> 00:16:48,000
postulated there must be a
restriction enzyme that was doing

207
00:16:48,100 --> 00:16:53,000
this restricting of phage growth.
When they found out what was doing

208
00:16:53,100 --> 00:16:57,000
it, they had discovered magic
scissors that would cut DNA at

209
00:16:57,100 --> 00:17:01,000
particular sequences.
So, the point here again,

210
00:17:01,100 --> 00:17:04,000
to try and go back and do this,
I hope some of you will get this

211
00:17:04,099 --> 00:17:08,000
anyway, many of the really important
discoveries come out of basic

212
00:17:08,099 --> 00:17:11,000
research.  They are easy to ridicule.
Why would I spend money on cancer,

213
00:17:11,099 --> 00:17:14,000
human disease or something, for
somebody studying some little weird

214
00:17:14,099 --> 00:17:18,000
phenomenon about phage.
But if you want to trace back to

215
00:17:18,099 --> 00:17:21,000
the experiment,
it sort of started the biotech

216
00:17:21,099 --> 00:17:24,000
industry.  It was the discovery of
restriction enzymes.

217
00:17:24,099 --> 00:17:28,000
It took a little while to discover
what it was, but the reason they

218
00:17:28,099 --> 00:17:31,000
were discovered,
were people were trying to

219
00:17:31,100 --> 00:17:34,000
understand that phenomenology.
Once we got restriction enzymes,

220
00:17:34,100 --> 00:17:38,000
we already had ligase, which is sort
of the tape we'd need,

221
00:17:38,100 --> 00:17:42,000
and just to show you here,
when we go back to this one,

222
00:17:42,100 --> 00:17:46,000
you can see that if we put these
together again,

223
00:17:46,100 --> 00:17:50,000
we have a three prime hydroxyl,
and a five-prime phosphate.  That's

224
00:17:50,100 --> 00:17:54,000
what DNA ligase knows how to do,
because that's how you seal up the

225
00:17:54,100 --> 00:17:58,000
end of a Okazaki fragment.
So, that particular part of the

226
00:17:58,100 --> 00:18:02,000
molecular biology toolkit was
already known to molecular

227
00:18:02,100 --> 00:18:06,000
biologists who had been studying
DNA replication.

228
00:18:06,100 --> 00:18:13,000
OK, so, if we took some DNA from
anything, and we cut up into pieces

229
00:18:13,100 --> 00:18:21,000
like this, and then we join them
with a vector that had been cut,

230
00:18:21,100 --> 00:18:29,000
so let's just sort of open it up a
little bit, this fragment would go

231
00:18:29,100 --> 00:18:37,000
in here into one vector
molecule.

232
00:18:37,100 --> 00:18:42,000
This fragment would insert in
another vector molecule,

233
00:18:42,100 --> 00:18:47,000
and so on and so forth.  Then we
would have what I said was a library,

234
00:18:47,100 --> 00:18:52,000
and the problem at this point is,
so you transform those into E. coli,

235
00:18:52,100 --> 00:18:58,000
and now we have a whole series of E.
coli.  They have their own

236
00:18:58,100 --> 00:19:03,000
chromosome, every one of them
because they still have

237
00:19:03,100 --> 00:19:10,000
to be a bacterium.
So, let's take three members of E.

238
00:19:10,100 --> 00:19:19,000
coli from this library, and they
will all have this vector,

239
00:19:19,100 --> 00:19:28,000
but they'll have, let's say,
insert number 1, 2, 3.  This insert

240
00:19:28,100 --> 00:19:35,000
is a little.
This insert is bigger,

241
00:19:35,100 --> 00:19:40,000
and so on.  If we did it right.
We have every possible fragment of

242
00:19:40,100 --> 00:19:46,000
DNA from the original source sitting
in its own vector.

243
00:19:46,100 --> 00:19:51,000
And the whole collection of E.
coli in this population in the

244
00:19:51,100 --> 00:19:56,000
certain library,
and the next part of the trick was,

245
00:19:56,100 --> 00:20:02,000
how do you find the thing you want,
especially if you take my DNA with 3

246
00:20:02,100 --> 00:20:07,000
billion base pairs.
That's an awful lot of restriction

247
00:20:07,100 --> 00:20:11,000
fragments no matter what you do.
How do you go about doing it?  So

248
00:20:11,100 --> 00:20:15,000
the experiment that I showed you at
the end of the lecture,

249
00:20:15,100 --> 00:20:20,000
cloning by complementation,
is fairly simple, and it was

250
00:20:20,100 --> 00:20:24,000
basically one of the first methods
that was used to find genes in

251
00:20:24,100 --> 00:20:28,000
recombinant library.
And that would be, for example,

252
00:20:28,100 --> 00:20:33,000
something that had thisGene mutation
in the chromosomal DNA.

253
00:20:33,100 --> 00:20:38,000
This is what the situation I'd
described the other day.

254
00:20:38,100 --> 00:20:43,000
So, if we put the library into
every cell, such that the bacterium

255
00:20:43,100 --> 00:20:48,000
we transformed the library into was
broken for the hisG gene,

256
00:20:48,100 --> 00:20:53,000
and that mutant couldn't grow on
minimal medium unless we put in

257
00:20:53,100 --> 00:20:58,000
added histadine.
But, if one member of that library

258
00:20:58,100 --> 00:21:03,000
had the wild type,
hisG gene, let's say it was this one

259
00:21:03,100 --> 00:21:08,000
here, maybe it had several
genes on it.

260
00:21:08,100 --> 00:21:13,000
But let's say over here.
We had hisG+, then the strain is

261
00:21:13,100 --> 00:21:19,000
back to being able to synthesize
histidine because it's got all the

262
00:21:19,100 --> 00:21:25,000
enzymes.  What I was pointing out
was this really is complementation,

263
00:21:25,100 --> 00:21:31,000
just like we did in that phage cross.
We've got one broken copy of the

264
00:21:31,100 --> 00:21:36,000
gene.  We've got a good copy.
And all you need is one good copy,

265
00:21:36,100 --> 00:21:41,000
and you're back in business.  What I
was saying at the end of the lecture

266
00:21:41,100 --> 00:21:46,000
was this is not a general solution,
though.    If I wanted to find the

267
00:21:46,100 --> 00:21:51,000
corresponding histidine gene from my
DNA, and all of these biosynthetic

268
00:21:51,100 --> 00:21:56,000
pathways, pretty much they rose so
early in evolution,

269
00:21:56,100 --> 00:22:01,000
the biochemistry is essentially
identical in all cells.

270
00:22:01,100 --> 00:22:06,000
Can I use this approach to find the
same gene for my DNA?

271
00:22:06,100 --> 00:22:09,000
What do you think?
Why don't you turn to somebody

272
00:22:09,100 --> 00:22:13,000
beside you and see if you can talk
for a minute, and then let's see if,

273
00:22:13,100 --> 00:22:17,000
I can think of at least a couple of
problems.  Let's see if you can come

274
00:22:17,100 --> 00:22:21,000
up with one or two of them.
Find somebody near you and see if

275
00:22:21,100 --> 00:23:06,000
you can come up with anything.

276
00:23:06,100 --> 00:23:14,000
Anybody want to volunteer?
An idea of why it would not work?

277
00:23:14,100 --> 00:23:22,000
Or some of you think it would?  No
ideas?  God, it's Monday.

278
00:23:22,100 --> 00:23:30,000
[LAUGHTER]  I feel like most of you
guys.

279
00:23:30,100 --> 00:23:36,000
Somebody, come on.
What do you think?

280
00:23:36,100 --> 00:23:42,000
It's going to work?
No idea?  What has to happen for it

281
00:23:42,100 --> 00:23:48,000
to work?  I'll give you a vector
that has my gene corresponding to

282
00:23:48,100 --> 00:23:54,000
that enzyme.  It's in E.
coli.  I need to make the protein.

283
00:23:54,100 --> 00:24:00,000
Yeah?  No, it's in the vector.  We
cloned it into an E.

284
00:24:00,100 --> 00:24:07,000
coli vector.  So, that's got it.
Yeah?

285
00:24:07,100 --> 00:24:11,000
Well it'll have a language that will
say "start transcription",

286
00:24:11,100 --> 00:24:15,000
but whose language is it going to
have?  It's going to have my

287
00:24:15,100 --> 00:24:19,000
transcriptional stuff.
Will that work in E. coli?

288
00:24:19,100 --> 00:24:23,000
Even though the open reading frame
is fine, that's good.

289
00:24:23,100 --> 00:24:27,000
How about translation?
I didn't even tell you about that.

290
00:24:27,100 --> 00:24:31,000
There is actually some specific

291
00:24:31,100 --> 00:24:35,000
stuff needed.  That's not universal,
either.  So when you get the RNA you

292
00:24:35,100 --> 00:24:39,000
still have to translate it.
There is another thing that might

293
00:24:39,100 --> 00:24:43,000
mess us up.  Do you remember
anything else about,

294
00:24:43,100 --> 00:24:47,000
yeah?  Introns and exons?
What if my gene has introns in it,

295
00:24:47,100 --> 00:24:51,000
which it almost surely has?  We have
to get rid of those.

296
00:24:51,100 --> 00:24:55,000
E. coli doesn't know what they are.
It's not used to taking them out.

297
00:24:55,100 --> 00:24:59,000
You see the issues?
Although that's a cute thing and

298
00:24:59,100 --> 00:25:03,000
that helps you find a gene from E.
coli by complementing E. coli mutant,

299
00:25:03,100 --> 00:25:07,000
or maybe you could do it with yeast
if you had a factor that would

300
00:25:07,100 --> 00:25:11,000
replicate in yeast, it wasn't
a general solution.

301
00:25:11,100 --> 00:25:14,000
So people had to use a whole variety
of different ways.

302
00:25:14,100 --> 00:25:17,000
Here's another way.
You know that you have that genetic

303
00:25:17,100 --> 00:25:21,000
code.  That was worked out years ago.
So let's say I was a biochemist,

304
00:25:21,100 --> 00:25:24,000
and I'd found a protein that I was
interested in,

305
00:25:24,100 --> 00:25:28,000
and I purified it,
and they got it out to single

306
00:25:28,100 --> 00:25:31,000
protein, and then I could cut it up
with things that proteases that will

307
00:25:31,100 --> 00:25:35,000
cut the protein into pieces,
and there are ways of sequencing

308
00:25:35,100 --> 00:25:39,000
protein.
I'm not going to tell you how it

309
00:25:39,100 --> 00:25:43,000
works in this course.
We just don't have time.

310
00:25:43,100 --> 00:25:47,000
But you can get the sequence of
little pieces of protein.

311
00:25:47,100 --> 00:25:52,000
And let's imagine that this was the
sequence of part of the protein that

312
00:25:52,100 --> 00:25:56,000
I purified.  It's one of my enzymes
[SOUND OFF/THEN ON] and I'd like to

313
00:25:56,100 --> 00:26:00,000
find the gene.
Well, how could I use that

314
00:26:00,100 --> 00:26:05,000
information to figure out where the
gene is in this library?

315
00:26:05,100 --> 00:26:11,000
So here's the strategy.
We get out the genetic code,

316
00:26:11,100 --> 00:26:18,000
which Gobind Khorana and Marshall
Nirenberg helped work out,

317
00:26:18,100 --> 00:26:24,000
and we say OK, alanine, and if you
look it up, what you'll find is that

318
00:26:24,100 --> 00:26:31,000
it's GC, and then it can be A T C or
G.  It can be any of those.

319
00:26:31,100 --> 00:26:36,000
If we look up what the codon for
aspartate.  We'll find that there's

320
00:26:36,100 --> 00:26:41,000
a G or an A, but it could be T,
or it could be C.  We look up

321
00:26:41,100 --> 00:26:46,000
lysine; it'll mostly be A,
but it could be A or G.  You'll

322
00:26:46,100 --> 00:26:51,000
notice the variation of those things
is almost all in  the third codon if

323
00:26:51,100 --> 00:26:57,000
that hadn't struck you.
Same thing with threonine: A,

324
00:26:57,100 --> 00:27:02,000
C, and this is another one;
therefore codons that encode this.

325
00:27:02,100 --> 00:27:09,000
And this one asparagine is that.
So knowing that piece of the protein

326
00:27:09,100 --> 00:27:18,000
doesn't define a unique sequence.
Though, what we could do it is we

327
00:27:18,100 --> 00:27:27,000
could synthesize what's
called a mixed probe.

328
00:27:27,100 --> 00:27:32,000
And that would mean when we are
going to synthesize this DNA and

329
00:27:32,100 --> 00:27:38,000
we'd start with a G building block.
And then we'd add a C building

330
00:27:38,100 --> 00:27:43,000
block.  So, now we made G and C.
And at the next step, we'd add an

331
00:27:43,100 --> 00:27:49,000
equal mixture of A,
T, G, and C.  So, what we would get

332
00:27:49,100 --> 00:27:54,000
out of that would be we'd be getting
G, and then the next biosynthetic

333
00:27:54,100 --> 00:28:00,000
step would give us G,
C.  And then the next biochemical

334
00:28:00,100 --> 00:28:06,000
step would give us GCA,
GCT, GCC, or GCG.

335
00:28:06,100 --> 00:28:10,000
At the next step,
we'd add a G.  So everyone of these

336
00:28:10,100 --> 00:28:15,000
would get a G.
Everyone would get an A,

337
00:28:15,100 --> 00:28:20,000
and then the next step, they would
branch.  And if you follow that out,

338
00:28:20,100 --> 00:28:25,000
you'll see by the end you have a
mixture of probes.

339
00:28:25,100 --> 00:28:30,000
One of them is going to be the
right one that you find in the DNA.

340
00:28:30,100 --> 00:28:34,000
Now if you work out the number of
possibilities,

341
00:28:34,100 --> 00:28:38,000
you'll discover that most of the
time there is only going to be one

342
00:28:38,100 --> 00:28:42,000
probe that's unique.
Once you get to about 20

343
00:28:42,100 --> 00:28:46,000
nucleotides, any sequence,
on average, is represented once in

344
00:28:46,100 --> 00:28:50,000
the human genome.
So as long as you make the probe

345
00:28:50,100 --> 00:28:54,000
long enough, one of the things in
your mixture will be a defined probe.

346
00:28:54,100 --> 00:28:58,000
So, what we can take is we have all
these different pieces of DNA that

347
00:28:58,100 --> 00:29:03,000
are the logical variants
you can see here.

348
00:29:03,100 --> 00:29:08,000
And then we would label the probe
with P32.  It's a radioactive

349
00:29:08,100 --> 00:29:14,000
isotope, and it's very easy.
You can add it to a five-prime

350
00:29:14,100 --> 00:29:19,000
phosphate.  There's a special enzyme
that will very easily take the

351
00:29:19,100 --> 00:29:25,000
terminal phosphate from ATP and put
it over.  It doesn't really [SOUND

352
00:29:25,100 --> 00:29:31,000
OFF/THEN ON] for this course
how we get it there.

353
00:29:31,100 --> 00:29:36,000
But we can do is radioactively label
the probe.  So now we've got this

354
00:29:36,100 --> 00:29:41,000
mixture.  And,
somewhere in this library is a piece

355
00:29:41,100 --> 00:29:46,000
of DNA that's going to have the gene
that's encoding the protein that

356
00:29:46,100 --> 00:29:51,000
we're interested in.
So, how do you go about trying to

357
00:29:51,100 --> 00:29:56,000
deal with that?
So, what we'll do is we'll plate

358
00:29:56,100 --> 00:30:01,000
our E. coli library onto a
bunch of Petri plates.

359
00:30:01,100 --> 00:30:07,000
So, I won't put too many colonies on
here, so we can sort of see a

360
00:30:07,100 --> 00:30:13,000
pattern.  But,
we'd have probably a lot of them,

361
00:30:13,100 --> 00:30:20,000
and we'd have a bunch of plates.
You can work out statistically how

362
00:30:20,100 --> 00:30:26,000
many plates you have to have to have
a chance of finding your gene of

363
00:30:26,100 --> 00:30:33,000
interest.  Then what we do,
is lay a membrane on the plate.

364
00:30:33,100 --> 00:30:40,000
It's a particular type of membrane,
and what that will do is it will

365
00:30:40,100 --> 00:30:48,000
make a copy of everything that's
there, and we're going to save the

366
00:30:48,100 --> 00:30:55,000
plate.  And then we're going to
treat the membrane,

367
00:30:55,100 --> 00:31:03,000
OK?  We've got a membrane that's got
an identical pattern.

368
00:31:03,100 --> 00:31:09,000
They've got some of the bacteria
from the colonies stuck at the

369
00:31:09,100 --> 00:31:15,000
corresponding parts on the membrane.
We're going to lyse the E. coli;

370
00:31:15,100 --> 00:31:22,000
that means break them open so all
their insides spill out.

371
00:31:22,100 --> 00:31:28,000
We will denature the DNA by
treating with a condition.

372
00:31:28,100 --> 00:31:35,000
You can, for example, vary the pH
and make the strands come apart.

373
00:31:35,100 --> 00:31:40,000
That gives single stranded DNA,
"ss" I'm using as an abbreviation

374
00:31:40,100 --> 00:31:46,000
for single strand of the strands you
pulled apart.  And,

375
00:31:46,100 --> 00:31:51,000
this sticks to the membrane.
So, now we've got, at every one of

376
00:31:51,100 --> 00:31:57,000
these little positions on the
membrane something that

377
00:31:57,100 --> 00:32:03,000
looks like this.
Here's the membrane,

378
00:32:03,100 --> 00:32:09,000
and there's some sort of single
stranded DNA that's stuck to the

379
00:32:09,100 --> 00:32:15,000
membrane in that fashion.
The DNA that's stuck here came from

380
00:32:15,100 --> 00:32:21,000
the bacterium here that had a
particular insert.

381
00:32:21,100 --> 00:32:27,000
Over here, we have all the E.
coli DNA and the vector DNA, but we

382
00:32:27,100 --> 00:32:34,000
will have a different piece of DNA
in the vector.

383
00:32:34,100 --> 00:32:38,000
Everybody with me?
OK, so if we were now to take our

384
00:32:38,100 --> 00:32:42,000
radioactive probe that we made up
there and get the conditions just

385
00:32:42,100 --> 00:32:46,000
right, that single stranded probe
will come in, and it will try and

386
00:32:46,100 --> 00:32:50,000
find its complement.
It'll form hydrogen bonds because

387
00:32:50,100 --> 00:32:54,000
that's the lowest energy well.
And, we think about it

388
00:32:54,100 --> 00:32:58,000
thermodynamically.
And, if we can get it right,

389
00:32:58,100 --> 00:33:02,000
the temperature and the conditions
right, nothing will stick unless

390
00:33:02,100 --> 00:33:06,000
it's an exact match
to the sequence.

391
00:33:06,100 --> 00:33:12,000
And, if we get the right probe that
can form hydrogen bonds with

392
00:33:12,100 --> 00:33:19,000
everything on here,
and it's got P32 at this point,

393
00:33:19,100 --> 00:33:25,000
what will happen is we'll have now,
the probe will stick, say, to this

394
00:33:25,100 --> 00:33:32,000
particular colony,
now with radioactivity right there.

395
00:33:32,100 --> 00:33:40,000
So, put some photographic film over

396
00:33:40,100 --> 00:33:48,000
the membrane, and right here there's
P32, and that'll expose the film and

397
00:33:48,100 --> 00:33:57,000
nowhere else.  And then,
when we develop, what we'll find is

398
00:33:57,100 --> 00:34:06,000
one, if this works well,
anyway, one spot.

399
00:34:06,100 --> 00:34:10,000
So, now we know that that piece,
that colony had a piece of human DNA

400
00:34:10,100 --> 00:34:14,000
in it that was related to the
sequence from the protein that I had

401
00:34:14,100 --> 00:34:18,000
purified.  So we go back to this
colony, I think our things have

402
00:34:18,100 --> 00:34:22,000
probably migrated around a little
bit here.  Let's move this up just a

403
00:34:22,100 --> 00:34:27,000
little bit, and make
it a little better.

404
00:34:27,100 --> 00:34:32,000
So this one is this one.
So, now I can go back to this

405
00:34:32,100 --> 00:34:37,000
colony and pick it out.
And, let's say it's this insert.

406
00:34:37,100 --> 00:34:43,000
So, now I've found I can sequence
the rest of that piece of DNA.

407
00:34:43,100 --> 00:34:48,000
We'll talk about how we sequence
DNA in the next lecture.

408
00:34:48,100 --> 00:34:54,000
So that's an alternative way of
identifying a clone of interest.

409
00:34:54,100 --> 00:34:59,000
There was a particularly painful
way of finding a gene in a library

410
00:34:59,100 --> 00:35:05,000
that we for the most part do not
have to do any more.

411
00:35:05,100 --> 00:35:18,000
It was called positional cloning.
And for example the gene that when

412
00:35:18,100 --> 00:35:32,000
it's broken causes cystic fibrosis,
it's a very difficult disease.

413
00:35:32,100 --> 00:35:37,000
Humans who have cystic fibrosis
have a very tough time.

414
00:35:37,100 --> 00:35:42,000
So there's a great deal of interest
in finding the gene that was broken

415
00:35:42,100 --> 00:35:47,000
in these patients.
Human geneticists,

416
00:35:47,100 --> 00:35:53,000
I showed you something about
pedigrees, they would have a

417
00:35:53,100 --> 00:35:58,000
chromosome.  They might have banding
patterns, and they would have

418
00:35:58,100 --> 00:36:03,000
figured out that somewhere along the
chromosome that the gene for cystic

419
00:36:03,100 --> 00:36:09,000
fibrosis lay somewhere between two
genetic markers that they

420
00:36:09,100 --> 00:36:13,000
had identified.
Now, the amount of DNA between

421
00:36:13,100 --> 00:36:17,000
something that you knew that the
gene was here,

422
00:36:17,100 --> 00:36:20,000
and something knew that the gene was
there, could be huge.

423
00:36:20,100 --> 00:36:24,000
It could be many, many,
many times the size of the E.

424
00:36:24,100 --> 00:36:27,000
coli chromosome.  So, what people
would do is they'd clone something

425
00:36:27,100 --> 00:36:31,000
from here, a little piece of DNA
from there, and also clone something

426
00:36:31,100 --> 00:36:35,000
and they get a little piece
of DNA from there.

427
00:36:35,100 --> 00:36:39,000
And then they go into the library,
and they try and find something that

428
00:36:39,100 --> 00:36:44,000
had this DNA and something that
extended in this direction the

429
00:36:44,100 --> 00:36:49,000
little bit.  By the sort of thing
you'd have marker A somewhere in the

430
00:36:49,100 --> 00:36:53,000
middle of the cystic fibrosis gene
but you didn't know exactly where.

431
00:36:53,100 --> 00:36:58,000
You'd clone a little piece of DNA
and use that to find another one

432
00:36:58,100 --> 00:37:03,000
that overlapped with it.
And that you'd find,

433
00:37:03,100 --> 00:37:07,000
use that to find another piece of
DNA.  You'd walk your way over this

434
00:37:07,100 --> 00:37:11,000
way, and you'd start the same
process at the other end.

435
00:37:11,100 --> 00:37:15,000
And, every one of these things,
the same kind of operation that

436
00:37:15,100 --> 00:37:19,000
we've got here,
so cycles, and cycles,

437
00:37:19,100 --> 00:37:23,000
and cycles of acquiring the next
adjacent piece of DNA,

438
00:37:23,100 --> 00:37:27,000
and working your way along here.
And you had to use more than one

439
00:37:27,100 --> 00:37:31,000
different restriction enzymes,
otherwise you wouldn't be able to

440
00:37:31,100 --> 00:37:35,000
get these overlaps.
And by doing that,

441
00:37:35,100 --> 00:37:39,000
eventually they were able to get all
the DNA that was between these

442
00:37:39,100 --> 00:37:43,000
markers.  They knew the cystic
fibrosis gene was there from the

443
00:37:43,100 --> 00:37:47,000
maps they had made by studying human
pedigrees.  And then,

444
00:37:47,100 --> 00:37:51,000
once you knew the sequence,
then you'd take candidate genes,

445
00:37:51,100 --> 00:37:55,000
and you'd take a bunch of cystic
fibrosis patients,

446
00:37:55,100 --> 00:37:59,000
and you'd start to see if every
person who had cystic fibrosis had a

447
00:37:59,100 --> 00:38:04,000
mutation in that gene.
And, eventually they got it.

448
00:38:04,100 --> 00:38:10,000
That process in the case of cystic
fibrosis took five years to do that

449
00:38:10,100 --> 00:38:16,000
with a huge team of people.
And, I guess it was from 1985-1990.

450
00:38:16,100 --> 00:38:21,000
If you wanted to do that experiment
today.  We come now to one of the

451
00:38:21,100 --> 00:38:27,000
most widely used ways to finding a
gene of interest,

452
00:38:27,100 --> 00:38:31,000
and that is you go to the computer.
The whole human genome sequence is

453
00:38:31,100 --> 00:38:35,000
in there.  If we were to do that
experiment today,

454
00:38:35,100 --> 00:38:39,000
we'd say, well, I know what this
gene is, so you look that gene up in

455
00:38:39,100 --> 00:38:43,000
the database.  And you knew what
this gene was and you look up the

456
00:38:43,100 --> 00:38:46,000
database.  Then you just look at all
the DNA that's in the middle.

457
00:38:46,100 --> 00:38:50,000
And you'd see a whole series of
open reading frames.

458
00:38:50,100 --> 00:38:54,000
And you'd probably say,
well, what do I know about the

459
00:38:54,100 --> 00:38:58,000
biology of cystic fibrosis?
Could I make a guess?  Is it a

460
00:38:58,100 --> 00:39:02,000
membrane protein?  Is it
not a membrane protein?

461
00:39:02,100 --> 00:39:05,000
And, there are certain
characteristics that would probably

462
00:39:05,100 --> 00:39:09,000
allow you to make a guess.
And then, you could jump right in

463
00:39:09,100 --> 00:39:13,000
and start sequencing DNA.
You could start the experiment

464
00:39:13,100 --> 00:39:16,000
practically that afternoon instead
of five years later.

465
00:39:16,100 --> 00:39:20,000
So, if you look back from the
literature, you'll find some of the

466
00:39:20,100 --> 00:39:24,000
key genes; in fact,
in human biology, we're isolated by

467
00:39:24,100 --> 00:39:27,000
this very painful process of
positional cloning,

468
00:39:27,100 --> 00:39:31,000
and you hardly ever have to do that
now.  It may be the odd case where

469
00:39:31,100 --> 00:39:35,000
something's needed,
but most of the stuff now,

470
00:39:35,100 --> 00:39:39,000
there are these amazing databases,
and I'll give you the URL for it at

471
00:39:39,100 --> 00:39:43,000
the beginning of next lecture.
And I'll show you when,

472
00:39:43,100 --> 00:39:48,000
this is an experiment in which
someone took this gene for cystic

473
00:39:48,100 --> 00:39:53,000
fibrosis.  It is a membrane protein,
and it's one of those proteins that

474
00:39:53,100 --> 00:39:57,000
mediates the passage of chloride
ions across the membrane.

475
00:39:57,100 --> 00:40:02,000
And, if that gets broken,
you end up with cystic fibrosis.

476
00:40:02,100 --> 00:40:07,000
What someone has done here is
they've taken that green fluorescent

477
00:40:07,100 --> 00:40:12,000
protein gene.
And, they've fused it to the end of

478
00:40:12,100 --> 00:40:16,000
the cystic fibrosis gene.
So, you can tell where the cystic

479
00:40:16,100 --> 00:40:21,000
fibrosis gene is localized in a lung
cell by looking to see where the

480
00:40:21,100 --> 00:40:26,000
fluorescence is.
And, I think you can see that the

481
00:40:26,100 --> 00:40:31,000
fluorescence is out there
along the membrane.

482
00:40:31,100 --> 00:40:35,000
OK, so at the beginning of next
lecture, I'll introduce you to how

483
00:40:35,100 --> 00:40:40,000
we take one of these recombinant
plasmids, and make what's called a

484
00:40:40,100 --> 00:40:44,000
restriction map.
It's using a very simple,

485
00:40:44,100 --> 00:40:49,000
little piece of apparatus like that,
and we'll go in and tell you about

486
00:40:49,100 --> 00:40:53,000
DNA sequencing,
and this PCR technique you've heard,

487
00:40:53,100 --> 00:40:56,000
preliminary chain reaction that
you've heard so much about, OK?