OK, so I'd like to go now to the next segment of the course.
Think you can probably appreciate little bit better this triangle I
had on before about how what biochemists did was they tended to
break cells open, look at the component parts,
through other things in there, and then proteins.
But an awful lot of stuff having to do with function is proteins,
and what geneticists, the discipline of genetics would do,
which made mutants of living organisms, and that looked at how
function was affected by mutating individual genes,
how those were both very powerful approaches. Genetics told you what
was really important, and biochemistry told you how it
worked at a molecular level, but the real problem is knowing
whether this thing you had doing something in the test tube was
actually the one that did it in life.
And I think it'll do when Arthur Kornberg isolated the very first DNA
polymerase, he was able to copy DNA. And he got a Nobel prize, and it
was the first enzyme that could copy DNA, and then John Karens made a
mutant that was lacking the enzyme. And the organism was still alive.
So therefore, it couldn't have been the DNA polymerase that was copying
the chromosome. It actually turned out to be a DNA
repair enzyme. So, if you can actually unite
genetics and biochemistry, if you can take a mutant that's
broken in a function and you found your protein was missing,
or vice versa, then you had a very, very powerful insight because you
connect your knowledge of what was physiologically important to the
biochemistry that you're doing. But it was really, really hard for
years, and only in very rare occasions did some geneticists have
a mutant that suggests so strongly that some biochemists would look,
or some biochemists would have such a powerful result that they talked
to a geneticist and seen if anybody had found the result.
And the power of recombinant DNA, although it started a biotech
industry, and it made possible the sequencing of the genome,
there's another level up one higher in conceptual understanding.
And what it did was, it let you go back and forth between here and here.
You wouldn't have any problem now if I gave you the sequencing of the
gene, you can order it. You could stick it in a cell and
make massive amounts of the protein. You purified it twenty-fold and it
would be pure, whereas before you might have had to
purified it fifteen thousand-fold out of 1,000 g of cells,
and you would have had to been a very good biochemists with 15 steps
in order to purify it. So, you can go from the sequence of
the gene to the protein, or if we got a protein and we wanted
to know which gene we'd just sequence a bit of the protein,
use that genetic code, work ourselves backwards to some possible
sequences, then go looking for the sequences, and then go
find the gene. So, what recombinant DNA allows us
to do is close that loop. You can go from genetics to
biochemistry at back and forth. Now everybody does everything
instead of it being isolated disciplines, which it was when I
entered the field. So, all of the stuff depended on
the development to clone particular pieces of DNA.
And I want to make clear right at the beginning,
there's a couple of uses of cloning that are in popular
usage right now. What we're talking about in this
lecture is cloning a piece of DNA. What that means, is I'm going to
take a particular segment of DNA, say, cut it here, and cut it there.
And I'm going to take that piece, and I'm going to do something to it
that lets me amplify it and make many, many copies of that piece of
DNA. And cloning of anything else you make a whole lot of copies.
So that's one use of the word cloning.
The other use, which you see in the popular press
all the time, is cloning an individual, but not being there but
you would take the nucleus from the cell of the individual,
he put that nucleus into an egg that didn't have its own nucleus moved,
and now you hope what you get out of that is an organism that has all the
same genetic content as the starting individual.
And in fact, although it sounds very good in paper,
is you're probably beginning to see it's not the panacea that people
thought it was, or that we'd have to worry that in
10 years, all of my MIT students would be clones of the brightest
person in the class or anything like that, because other stuff happens,
because unless you go on to advanced biology courses,
but there are modifications of DNA. There's all sorts of stuff that
happens to it, so it's not identical.
And so many of these cloned organisms, like Dolly the sheep,
that was famous, died early with, I forget, arthritis and things. So,
there are a lot of problems on that score. But that's the other use of
the word cloning and that's not what these next three lectures that we
are talking about cloning a piece of DNA. And that was the big problem
that faced the field, certainly when I was an undergrad
and even when I was a grad student I was interested in synthesizing
pieces of DNA, and it was one of those things that
people said, why are you doing it? Well, because you could try to do
it. What if you got a piece of DNA, like Gobind Khorana, who's my
colleague, who got the Nobel prize for synthesizing the first gene.
He synthesized it. It was a tRNA gene that was 120
nucleotide base pairs long or something. He synthesized it.
He'd shown you could do it. But you couldn't do anything with it.
And there were sort of two big problems. One was the fact that
this DNA, although it's not its a monotonous tetranucleotide.
It's pretty hard to tell. Each one of these things is a base
pair, and human DNA has 3 billion of those. And a bit down here,
doesn't look very different than the bit out there.
And it certainly wouldn't looks very different than the bit of DNA
that's 2 billion base pairs over on the other side of campus or
something. So, there is no way to take DNA and cut
it reproducibly, so you to get fragments.
What you could see from first principles was what you would need
was magic scissors. And what would the scissors look
like? Well, it would have to be scissors that could be sequenced
because there's nothing else different. You know it's a regular
backbone, and it's only four nucleotides. So,
if you wanted to cut DNA in particular places,
you had to have scissors that could see a sequence.
And furthermore, you can see they couldn't just,
there are the hydrogen bonding parts of ANT or GNC because those are
stuck together there in the middle of the DNA.
So, you'd need scissors that could somehow find a sequence and make a
cut. And those were found. I'm going to till you about those.
They're called restriction enzymes. So that was part of the thing. The
other thing was, imagine I could cut out this
fragment. And they gave it to you, and I said, great. Now I've got it.
Would you make me a lot of copies of this DNA?
Could you do that? Let's say, you now know how to
transform the principle that we saw back with Avery.
We could take naked DNA and put this fragment into the cell.
Would it replicate? What do you think? Anybody remember?
No? OK, we talked about some other languages, right?
But one of the things that's in the DNA is the genetic code with all the
genes. And we can find the reading frame. Remember when we talked
about an origin of replication. I said that was sort of, at least
for E. coli there's one origin. In eukaryotes the origins are
spaced out along the DNA. And every time you have a round of
replication, it starts with one of those origins and then goes.
So the chance of this piece of DNA by chance is going to have an origin
is pretty small. So if I put it into an organism,
it's going to sit there, if you're lucky, make it degraded because it's
got [blend ins? , or even if I made it into circle
it probably wouldn't replicate because it probably doesn't have the
word in the DNA that says start a round of DNA replication.
So, the other overarching principle of DNA replication is you somehow
have to take the fragment of DNA that you're looking at,
and you have to attach it to an origin of replication.
Now, if you have an origin of replication and you have a fragment
of DNA, and you put it in the cell, now you'll get a lot of copies of
that piece of DNA. So that's what recombinant DNA is
all about in a really, really simple form. I'm just going
to take you now into increasing sort of levels of detail.
So, let me just sort of give you just sort of a really broad view of
this cloning, and then we'll sort of start to dive in to some of the
fancier techniques that have come out of this.
We'll talk about DNA sequencing, and PCR, and stuff in the next
lecture. So the first principle here is to cut the DNA,
and I know you may think this is sort of baby talk,
but this is how I think. If you really think about this
stuff, this is what it really is. With sequence specific molecular
scissors, these have the rather odd name.
They're called restriction enzymes. I don't know if any of you know why
they're called restriction enzymes, and although I'm sure that some of
you have used them in your op to cut up pieces of DNA.
But what that does, these are enzymes, as I'll tell you,
that recognize a particular sequence. And they always cut at that
sequence. In the value of that is you can reproducibly cut DNA exactly
the same spot, and the spots are specified by
whatever sequence that particular pair of scissors knows how to read.
Then the next thing we have to do is we need to join the piece of DNA to
an origin of replication. So the thing that carries the
origin of replication is called a vector. And usually,
not always, these are circles. We'll consider the ones that grow
in E. coli are most of the time, or in bacteria, mostly circles.
They are the ones that are broadly used for most cloning.
So, we'll talk about those. What makes a vector? When it has
to have is an origin of DNA replication. They usually have
something else. We could call it a selectable
marker, but something like a drug resistance.
If any of you have done cloning in a UROP usually it's something like a
gene for making the cell ampicillin resistant, or tetracycline resistant,
some antibiotic that would normally kill the cell.
So you can tell, does that cell have that vector or
not because the cell starts at either ampicillin sensitive.
It acquires the vector that's replicating it,
also acquires the gene that gives it the drug resistance.
But if we're going to cut that, if we're going to join a piece of
DNA to that, we can't join into a circle without breaking it.
So, we need to cut the vector at a unique site. And we would use a
restriction enzyme for that. And you can also see in designing a
vector, you'd want something that only has one site.
So, what we would have achieved from this conceptually as we've now got
this, this is the vector, its origin of replication here,
and let's say ampicillin resistance, for example, as a selectable marker,
the gene for that could be encoded here. And we have the fragment of,
if you want to that down, just put it down on the floor,
I think. We've made the point at this stage. Thanks.
What one has to do is to join this piece of DNA to that.
And we'll go to the molecular details of this.
But, we'll join the fragment to the vector, and actually this was
something that was already in molecular biologists' toolkits,
have been studying DNA replication. That's DNA ligase.
When we finish an Okazaki fragment, we had to seal [UNINTELLIGIBLE] and
the enzyme that did that was an enzyme called DNA ligase.
So, molecular biologists basically had the scotch tape or the glue to
join stuff back together. What they were missing for many,
many years where the sequence specific molecular scissors.
So at this stage, if we were doing the recombinant DNA,
we now have a vector. We now have a piece of DNA joined
to it. In fact, we probably have a whole
other mess of things that happens along the way.
But at the moment, they're in a test tube.
So, if we want to have this thing grow, what do we have to do next?
We are going to have to get the DNA from outside the cell inside the
cell. That's the word; we need to transform the DNA into a cell.
Again, the word transform, that goes back to those
transformation experiments with the Streptococcus pneumonia going from
smooth to rough, and you are taking stuff from the
cell that transformed them from rough to smooth,
whatever, that's where the word came from, but we now know it's getting
naked DNA inside the cell where it can be replicated.
And then the next thing we need to know is, what cells have acquired
this vector that at least as the vector.
We'll settle for that in the beginning, and to do that,
you need to select for the marker on the vector. In the case of this one,
we would start with a strain that killed my ampicillin,
and then we just play it out and ask for guys that are ampicillin
resistant. And, you can see that there is
another class of problem because if we had uncut vector,
and there would probably be, for sure, some of that in our mix,
that would make the cells ampicillin. And if we had an insert,
it would also be ampicillin. So, if we really wanted to get into
this, we'd have to do some more work to sort out what's on there.
But that's the basic stuff. I suspect most of you know this
practically since kindergarten. But that's the overall framework
into which, now, I'm going to start layering
different pieces of detail. And the next part,
again, some of you may know. I don't think it will be a totally
foreign concept. You are probably familiar with this,
that what are these restriction enzymes? The actual word is
restriction endonuclease. They are often usually called
restriction enzymes in a lab [parlons?]. Nuclease is something
that cuts the nucleic acid, and endonuclease is one that doesn't
need a free end. So, it can cut in the middle of the
sequence instead of nibbling at the end. That would be
an exonuclease. So, these things have names that
tend to be something like ECO-R1, which has something to do with where
they are derived from. And a typical one, one of the very
first ones that is still in really wide use, is ECO-R1.
And this recognizes the sequence G A A T T C.
Now, you'll notice that if you read the sequence in this way it's the
same sequence when you read it on the other strand.
It's called a palindrome but be careful because palindromes in
English, those are words that you read from the front to the back;
they're the same. In an English letter,
it doesn't matter whether it's in A here or an A there.
But you guys know something about DNA structure.
There's a five to three prime polarity. So,
reading this way doesn't look at all the same. It's totally different.
But reading in this strand, we say that's five to three.
The thing that's identical is the reciprocal sequence on the other
side. So there is this, you see G A A and G A A but it's not
like the English word palindrome, so get yourself mixed up about that.
Anyway, what this will then, what this enzyme then does, is it
cuts to the side here. It cuts symmetrically.
And what it generates them is a G three prime hydroxyl.
Remember the ribose? If we have, say, an A there, this is the three
prime position, and that's the five prime position
in the sugar. So, it leaves a three prime hydroxyl,
and is also then leaves a five prime phosphate.
So, we'd have A A T T C here, and then on the other side, we would
get the reciprocal thing. So, we'd have G with a three prime
hydroxyl, and then over A A T T C like that.
So now we've got a break here. We can pull those apart. But one
of the nice things you can see right from this is that we're generating
five prime single stranded ends, and this one is the sequence A A T T
C. This is A A T T C here, and these guys, if they could get
together and line up as they would here, they'd be able to
form hydrogen bonds. So, if you take an enzyme like
ECO-R1, and we took, let's say, a circle that had a
single ECO-R1 site, G A A T T C, if we cut it with the
restriction enzyme we would make [nicks?]. And if we kept a cold,
all that we'd have is DNA nicks. And if we warm things up a little
bit, there's only four hydrogen bonds that are holding that together.
So, the thing would linearize and just flop around in the breeze.
If we cooled it slowly, the thermodynamically most favorable
state, the lowest energy state, would be with those ends coming back
together. So, we could then add DNA ligase.
If we added these up and added DNA ligase, we could reverse the process
and go back and forth, ECO-R1 to cut it, DNA to ligate it.
And then, the beauty of recombinant DNA is this rejoining part doesn't
see what's out here or what's out there.
All it sees is the little ends that are generated by an ECO-R1 site.
So they take some of my DNA, and I'll cut it up.
I'll get a zillion ECO-R1 fragments, but they'll all have the same little
overhanging bit that's complementary to the vector.
So if I take a vector cut with ECO-R1, and I take some of my own
DNA and I mix them, I can get a little fragment,
get in between the vector, and it does exactly that joining that I was
diagramming right here. So again, it was the discovery of
these restriction enzymes that made possible almost all the stuff that's
happened in biology since 1975. The development of restriction
enzymes was essentially, I was a postdoc at Berkeley at that
point and the labs, Stan Cohen at Stanford,
Herb Boyer at UCSF, and a two others around the country were
working on this. They were almost all labs that had
worked on bacterial plasmids. Plasmids are little circles of DNA,
so the labs that started were ones who have been busy studying little
circles of DNA that usually carry drug-resistances between cells.
And so that was happening while I was a postdoc.
And when I got to MIT and '76 the technology was just beginning.
I was one of the first labs trying to cut pieces of DNA and join
them back together. So, it's a pretty recent development.
At that point, DNA sequencing hadn't been invented.
The idea that you could pull out a piece of DNA and do something with
it or produce a protein was just a thought. It didn't exist.
So it's hard to overemphasize how critical the discovery of these
restriction enzymes were. Now, I just want to tell you where
they came from, or how people found them.
And they'll try and do this quickly because I know some of you get
impatient with history. But this is really important because
it's very easy to make fun of basic research. You can ridicule anything
pretty easily, and you might just ask because I'm
telling you the story. Somebody proposed doing this.
I'm going to tell you the experiment that basically is the
basis of the biotech industry, and would you have been smart enough
to recognize that it was the discovery of a phenomenon called
restriction, restriction in bacteriophage growth on bacteria?
And it was, here are actually a couple of EM's and these little
plasmids. This is an electron micrograph one.
In these little circles it's been shadowed. And this is actually
artificially colored, but that was the kind of plasmids
that people were cutting up. So, as I said, trying to get
through this DNA, and the stuff, what's made possible
the sequencing at the Whitehead Genome Center and stuff that I'll
tell you about is going on. I didn't really set this one up.
But that's Eric Lander who teaches 701 in the fall.
I told you a picture from that DNA 50th. Well, they had a banquet at
the end of it, and I was there.
This is [Savandi Pabo? from Europe who is sequencing the
chimp genome. And that's Francis Collins who is head of the entire
Human Genome Project. This is Evelyn Witkin,
who was a big discoverer of early DNA repair events.
And I put that one in because it was sort of interesting.
There was Eric, and Savandi, and Francis were
talking about what would happen when they knew the sequence of the chimp
genome, which wasn't done. And there was an advertisement,
a poster advertising Jim Watson's latest book. And they ripped that
in half, and were writing notes on the back all the way through dinner.
So if you want to see what scientists on the cutting edge,
including someone who teaches 701 the fall looks like when they are
not teaching 701, there is a picture.
So anyway, the discovery of restriction enzymes was Salvador
Luria, who I've mentioned. He was a member of the biology
department, and one of our Nobel Prize winners.
He started the cancer center. He also trained at Jim Watson,
when I showed you that picture. This is Salvador standing over here.
Another thing that Salvador did, he was a Nobel Prize winner but he
thought introductory biology. So I am basically following in the
footsteps of Salvador. He wrote a book called,
even though he was a Nobel Prize winner, a book called 36 lectures in
introductory biology. And some universities,
the intro to biology is taught by whoever is at the bottom of the food
chain. The most junior professor gets stuck with intro to biology.
And here it's the other way. I mean you're getting Eric and Bob,
for example, Weinberg to teach in the fall, tells you that.
And really where that comes from is the fact that Salvador Luria had
such an interest in replication. So he trained at Jim Watson,
started the cancer center here, and he also carried out this
phenomenon of restriction. And to get this working with
bacteriophage. And I know a [couple he wrote?
, he didn't like to see old guys on porches. So I got freaked out,
and I took this next picture out for this morning. Oh,
I've got to show it anyway for two reasons. This is Salvador sitting
on a porch at Cold Spring Harbor with Max Delbrook who started,
really, much of the work on bacteriophage that gave us the
underpinnings of microbiology. And then put it partly on A because
it shows the informality of the molecular biology culture which
persists to this day, and also because Salvador had such
an impish sense of humor. You would have really enjoyed had
he been teaching this course. Anyway, Salvador was studying this
bacteriophage. Remember we talked about it?
And they basically [with syringe? they injected their DNA into the
cell. There's an electron micrograph. The DNA is up top there.
It goes in, and then the DNA takes over the cell,
reprograms it, and makes babyphage. And I showed you how we make plaques.
So that was what Salvador was studying. And it's a little like
what we are talking about with Mendel. He didn't have very many
techniques available to him at the time. He couldn't sequence DNA.
He couldn't do a lot of things. But he could [plate phage?
and count, and things like that. And what Salvador was looking at,
he had a bacteriophage, and he had two strains of bacteria.
I'll call them A and B. OK, here comes the experiment that
founded the biotech industry. You ready? You going to fund me?
All right, so what I propose doing is I'm going to grow the phage on
strain A, and now I'm going to plate on strain A and strain B.
I laid awake all last week thinking of this experiment.
So what did I get? I got a lot of plaques on strain A,
probably something like 109 or 1010 per mil because that's what this
phage lysate usually looks like once you've grown them up.
And if I plate them over on strain B, no phage, maybe an
occasional phage. So, I bristly the bacteriophage can
grow mine on strain A. It can't grow on strain B most of
the time, but some variant has managed to figure out how to grow on
strain B. Give our foray into genetics, I think many of you would
think, probably as I suspect Salvador did at the time,
the things mutated. It's learned. It's made some change in its
genomes that's allowed it to grow on strain B. So,
I wonder if it learned to grow on strain B, couldn't
grow on strain A? So, basically what he took was the
phage from that experiment, and then he plated them on strain A
and strain B. And, well, as you might guess,
since it was growing on strain B, lots of plaques, and there were lots
of plaques over here. OK, so it didn't forget how to grow
on strain A. So, better check over here,
too, need a control experiment, so take this guy, plate it out,
strain A, strain B, some plaques over here.
That's not a surprise going in strain A.
We are back to where we started from. It doesn't sound like a mutant,
does it? And if it was a mutant, everybody should have been the same.
Instead, when you grew a phage on strain A, it didn't have the ability
to grow on strain B. But if you gave it a chance to grow
on strain B, most of them wouldn't make it.
But if it ever did, it had now acquired the ability to
grow on strain B. So it could still grow on both.
But if you take somebody who had been growing, something had been
growing at strain A, it lost it. And so, the idea there
was then that it wasn't a mutation. Something was happening in the
strain B that enabled it to grow on strain B.
And if it ever got away from that environment, [it?
lost it. So, the phenomenon was called restriction.
It wasn't a mutation. It was something else.
And it turned out, then, that restriction was due to an
enzyme. An example of this kind of thing,
then, would be this ECO-R1 activity that's able to cut at a very
specific sequence. Now, if you are going to have a set
of molecular scissors inside of you that could cut a G A T C sequences,
you'd have a problem unless you did something else because every one of
your G A T C sequences would be cut by the restriction enzymes.
So what the cells that have a restriction enzyme have,
as a modification enzyme that recognizes the same sequence,
and then modifies it in some way that makes it resistant to the
restriction enzyme. And in the case of the ECO-R1,
it puts on a methyl group on this A. You might have thought that that
would interfere with base pairing, but it doesn't because adenine looks
like this. These are the guys that do the base pair refining,
if you look back, and you'll see that you could put a methyl
group in there. It wouldn't interfere with the base
pairing, but would allow this to go. So, it was the discovery of this
phenomenon of restriction of bacteriophage grew on one strain,
not on another. It could learn to grow on the other strain.
It could lose that acquisition. It was that phenomenon. People,
didn't have any other reason other than it was an interesting problem
in biology to understand it. Once they understood the basis of it,
another whole world opened up because you could see from basic
principles, now I could cut any piece of DNA. I could generate
these little overhanging sticky ends. I could take a plasmid.
People knew about those. I could find one that only has one
restriction site. I could stick things in.
Now I've attached an origin of replication to each of those pieces.
And I'm in business. I can now, for the first time ever,
take a particular piece of DNA and make as many copies as I want.
And that was an absolute transformation to the way people
were able to think about biology. So I'm going to just kind of give
you an idea of how people would start. So the way people began and
still began most things is they'd call it, the usual term is you call
constructing a recombinant DNA library.
And there are a variety of different ways of doing this.
But this principle is the same. We'd take the DNA from whatever
organism you're interested in, [and studying?].
And we cut with some restriction enzyme. And this restriction enzyme
will cut wherever there happened to be sites. They might be close
together. They might be far apart. But whatever, still generate some
characteristic set of fragments. And we'll now have,
in this case, fragment number one, fragment number two, fragment number
three, fragment number four, and so on. And if it's my DNA,
there's a lot of fragments. And of course, they're all mixed up.
I can't tell where any of them are. They're just all mixed together in
the test tube. Then we'll take that vector that
we've opened. And now, we'll mix all of these
fragments together with this vector. And then we join it just the way
[that it's cut? . And now what we'll get,
it's a collection of plasmids that have different inserts.
So, one of the plasmids will have that fragment number one.
Another one of them will have fragment number two, number
three, and so on. Then this whole thing is what's
known as a library. You can see if it's DNA from me,
there were 3 billion base pairs to start. Given the human DNA,
G A T C sequences are pretty common. You can calculate the frequency
yourself for how many sites on average there would be for a
restriction enzyme within a piece of DNA and figure out roughly how the
fragments there would be in the library of human DNA.
So, we are partway there. We can now make a library.
We can make it from bacterial DNA. We can make it from human DNA. But
the next thing that people had to learn how to do was to figure out
how to find a particular fragment that had the gene that you
are interested in. And there's a whole variety of
things. I mean, ultimately today since the human
genome is sequenced, you go on a computer and a type and
you find it because the sequence is all known. But the only reason we
can do that is because of all the work that was done in between.
So, I'll give you several ways of doing this. But one of the ways I
think you can see very easily, and it's actually going back to the
term complementation. Remember complementation?
We had something that was mutant, and then we'd put in a wild type
gene, and fixed it up again. So, for example,
suppose I was studying histidine biosynthesis in E.
coli, and I wanted to find the gene that encoded the enzyme that I had
just disabled in my histidine minus mutant. So, if I have a
[hisoxotroph?], I'll call it a [his G?
gene, for example, that's one of the genes involved in making
histidine. So, since it's a histidine auxotroph,
if I have it on just minimal glucose plates, and I streak it out,
it's not going to grow. But if I grow it on minimal glucose
plus histidine, then it will be able to grow,
right? So, I've got a variant of this organism.
It's got a single mutation in it that's affecting one gene.
And because I don't have that gene, I can't grow on minimal.
If I made a library of E. coli DNA, which is going to have a
lot of fragments as well, and I took that library and I put it
into this mutant, I'm going to get a big mess of
things, all the different plasmids with all the different fragments
that go into that mutant. How am I going to find the one that
I want? Anybody see that? It's not that hard. I'm the mutant.
I can't grow on minimal because I can't make this enzyme.
Therefore, I can't make histidine. What do I need? How could you fix
me up if you were a doctor? What's the gene we want out of here?
The one that makes that particular enzyme that makes histidine.
Yeah, take the whole library, stick it in this mutant. If the
gene coming in encodes DNA polymerase, I'm not going
to help this guy. It still won't grow in minimal,
take a gene involved in making part of the cell wall would help.
But if I put in the, I get a fragment of DNA that includes the
his G plus gene, and I put it in here,
it's going to grow up. If it has the plasmid that has,
or let's say the vector that has the his G plus gene.
So what you've done is a really, sort of, you've used that principle
of complementation that some of you were sort of wondering about when we
were doing genetics. So, you'd break a copy of a gene.
In the things we talked about, we bring in a whole chromosome that
included in it just a wild type copy of the gene. With recombinant DNA,
we can really narrow it down in the extreme. We can bring in a piece of
DNA that is only the gene that's broken.
And we can take the gene back to the wild type. One thing,
just to close, you'll see, if you remember back when I talked
about language that are not universal, although the genetic code
is universal, promoters and things are not. So I couldn't ever do this
with a human DNA, could I, because it wouldn't get
expressed. So, we need some other ways of finding
those. We'll talk about those on the next lecture, OK?