ADAM MARTIN: And so I just want to say a couple sentences
about DNA sequencing, just to finish that up.
And so you'll remember this slide from last lecture.
And remember, the way this Sanger technique works
is to set up four different reactions where
each reaction has a different one
of these dideoxynucleotides.
OK, so there's four reactions, each
with different dideoxy NTP.
And I brought along a gel that I ran a while ago,
which is basically-- it's from sequencing gel, and you can--
I'll pass this around so you can take a look at it.
So the four different lanes for each sample
are the different dideoxynucleotide reactions.
And what I want you to notice as that's passing around
and you're looking at it is that the different reactions
with the different dideoxynucleotides
give different patterns of DNA fragment lengths.
So there are different patterns of fragment lengths.
And the different patterns are based on the fact--
this is based on the sequence, the sequence of the template,
OK?
And so if we look at the example up here, what you'll see
is that in this banding pattern for dideoxy TTP,
you see that there's a really short fragment at the bottom
there, and so that fragment indicates that there must be
an A in the template sequence.
The next fragment up would be this one in the dideoxy GTP
lane, and that indicates that one nucleotide beyond this A
is a C position, and so on and so forth,
such that you can sort of order the fragments
and see which reaction has a fragment
and then read off a DNA sequence.
OK, so conceptually, that's how you
would read off the sequence of a given strand of DNA, OK?
So you might be wondering, if now, we just read off
sequence as a series of colors, why am I even introducing
this technique?
And the reason is because I think
it's important for you as potentially future scientists
to know that when you're faced with a problem,
how you might discover something new.
And I see the Sanger method of DNA
sequencing as a really clever and elegant way
in which Fred Sanger solved the problem of DNA sequencing,
and while we don't necessarily do it that way today, it still
illustrates a concept that's important,
the concept of chain termination,
and I think there is something to be learned from this older
technique, even if it's not exactly how we
sequence DNA today.
So for today's lecture, we're going
to continue on our quest to basically clone a gene that's
responsible for a disease.
And so we started this in the last lecture.
And I guess one thing we would want
to start with is a disease, so I'm
going to introduce to you now a disease called aniridia.
And in order to clone the gene for a disease,
it has to be a heritable disease,
in this case, because we're going to use linkage analysis
to identify it.
So aniridia is a disease that's an eye disease in humans.
It's a rare eye disease.
So I want to show you a bit of an example of this eye disease.
The way this disease manifests itself
is it's basically the affected individual
has an eye that is lacking an iris.
So I'm going to show you what this looks like.
If you're squeamish or don't like weird eyes
and you don't want to look, you can look away.
But I will show you affected phenotype in 3, 2, 1, OK,
everyone looking who wants to see weird eyes.
OK, good.
So that is a individual that has aniridia, and also this one.
So you see there's no clear iris in these eyes.
And this disease is associated with other abnormalities
of the eye that severely impair vision.
And this is an inherited disease,
and this is a pedigree from a family
or series of families where the disease is propagating.
And so anyone have a suggestion as to what mode of inheritance
this is?
Anyone want to rule a mode out?
Rachel, you have an idea?
AUDIENCE: I was going to say X-linked dominant,
but [INAUDIBLE]
ADAM MARTIN: OK, so let's take X-linked dominant.
So if it was X-linked dominant, then this male
would have an X chromosome with the dominant allele
of the disease and should only pass it to his females.
So I don't think that it would necessarily
be X-linked dominant.
Anyone else have an idea?
Yeah, Georgia?
AUDIENCE: Autosomal.
ADAM MARTIN: Autosomal dominant.
I like autosomal dominant.
So in this case, you see you have
an individual with the disease and they
marry into a family with no history of the disease.
One thing I'll point out, for many of these diseases,
they're extremely rare, so if you see sort of a family tree
where there's no instance of the disease,
if it's a rare disease, it's likely
that these individuals are not carriers.
And so in this case, if you assume that this person doesn't
have any form of the--
isn't a carrier for the disease, then this
cross here resulting in about half
of the individuals affected with the disease, that
would be a characteristic of an autosomal dominant disease.
So everyone understand my logic?
Yes, Carlos?
AUDIENCE: What are-- why is two and that
looks like three on the slide, why are they crossed out?
ADAM MARTIN: I think they're deceased.
Yes.
OK, so let's say you have a pedigree.
You have pedigrees, you're able to try to link this marker to--
or the disease phenotype with various molecular markers,
which we discussed in last week's lectures,
then you're on the way to performing
a process which is known as positional gene cloning.
And what positional gene cloning is
is it's basically cloning a gene and a allele that's
responsible for a disease based on its position in the genome,
it's position in a particular chromosomal region.
So it's basically cloning a gene based
on its chromosomal position or its chromosome position.
And the first step of positional gene cloning
would be to establish maybe what chromosome it's on.
And a straightforward way to do this, as we've basically
been discussing almost from when I started lecturing,
is to create some sort of linkage
map or do linkage mapping to identify,
in the case of humans, molecular markers that this disease
allele is linked to.
And remember, in last week's lecture,
we talked about a number of different polymorphisms
that are present in the human genome
that we can use to establish linkage with a given phenotype.
In this case, it's a human disease.
And we talked about this example for a microsatellite marker.
And in this case, we talked through this example
of how this dominant allele, P, is linked
to this microsatellite allele m double prime,
because if you look at the pedigree here,
all of the affected individuals here
contain this m double prime sized fragment
for this microsatellite.
Another thing to notice here is you
can see that this couple has been faithful to each other,
because basically, each of the children
have an allele from the father and an allele from the mother.
So you can see that type of--
you can see that using this type of molecular marker as well.
OK, so you establish linkage.
So linkage mapping establishes the chromosome position
of a given allele and the gene.
And this chromosome position sort of
gets maybe in the right country, but you still
have a long way before you get to the specific street address.
And so you have to then sort of narrow it in
to identify a smaller region of the chromosome that could
possibly contain this gene.
And so what you would do is go from this linkage map, where
you maybe identify the position of this gene within a couple
map units, to this next resolution of map
called a physical map, OK?
So we go from the linkage position
to the physical map of the chromosome.
And the physical map, as the name implies,
is when you have physical pieces of DNA
that are present in this region of the chromosome.
So the physical map means you have
cloned, so recombinant pieces of DNA,
cloned pieces of DNA which encompass a given chromosome
region.
So these are encompassing a chromosome region.
OK, so how would you get a piece of DNA
that sort of is in this region?
How would you start?
How would you start fishing for that DNA?
So you've gone through the process of linkage,
you've identified sort of a polymorphism that is
linked to the disease allele.
How would you go from there to getting a physical piece of DNA
that is present in that region of the chromosome?
So let's think back to--
Jeremy, did you have an idea?
AUDIENCE: Start by using PCR to just amplify that chunk.
[INAUDIBLE]
ADAM MARTIN: And what primers, I guess,
would you use for the PCR?
AUDIENCE: Depending on which chunk you're trying to get,
you'd use [INAUDIBLE]
ADAM MARTIN: OK, so Jeremy is saying
if you knew the sequence, and I guess
if you're doing this microsatellite analysis,
you had primers that recognize a sequence at a given
genomic position, so you actually know something
about the sequence because of this polymorphism,
so you can use that knowledge to then look for this sequence.
And you could even look for the microsatellite in a DNA
library.
OK, so you have cloned pieces of DNA,
and you're going to start with--
I'm going to swap this.
Your starting position could be one
of these polymorphisms in the sequence around it,
which you already know.
So let's say you had this microsatellite marker.
You could then-- what I'm drawing here
is a piece of genomic DNA.
So this is genomic DNA.
I'm just drawing the insert.
This would be recombinant DNA.
It would be present in some vector or plasmid.
But if you can identify the sequence that
contains this microsatellite marker,
then you would have the microsatellite, but also
the surrounding DNA, OK?
So that sort of anchors you at a given position.
Now, you don't know if your gene is in that piece of DNA,
but you know that it's linked, and so it
should be around that piece of DNA somewhere.
And so it's unlikely your gene is
going to be on this small piece of DNA that's cloned.
This is probably just a few kb, and you could still
be very far away from this, but that
serves as a starting point from which you can go from
to get more and more pieces of DNA
such that eventually, you have a bunch of pieces of DNA
that are going to span the entire region.
So the way you identify other pieces of DNA is you
could start with a piece of DNA maybe at the end of this insert
and look for other inserts that are not
identical to this piece that also contain this piece here.
So that might get you a piece that's
overlapping, but extends farther than your initial piece.
So now you've moved slightly farther away
from your starting point, which is this starting polymorphism.
Then you could choose maybe another DNA sequence here
and look for a piece of DNA that, again,
is extending a bit farther out.
And so you can see how iteratively,
you can get farther and farther away from this starting point
that you know your gene is linked to.
And this process of going sort piece by piece and clone
by clone away from a starting position
is known as a chromosome walk.
And you can do this bidirectionally.
So you could also start with a sequence of DNA
here and look for a clone that goes the other way.
And you can see on my slide up there,
you can see that in this case, they've
taken a one map unit region of the chromosome
and they're illustrating physical pieces of DNA that
are overlapping that encompass this entire region.
So this could be much bigger than the amount of DNA that
would fit in one of these clones in the bacteria,
but by sort of identify overlapping clones,
you get the entire region.
And what this is called here, because these pieces of DNA
are contiguous with each other, this is known as a contig.
Yeah, Jeremy?
AUDIENCE: So would how you get the--
once you find one of those pieces,
how do you get the primer for the end of it to start?
Do you actually sequence each of these pieces of DNA?
ADAM MARTIN: You could sequence it,
or you could use a technique that I'm
going to talk about at the end of my lecture, which
I'll come back to.
So nowadays, you'd probably just sequence it and then maybe look
for that in another clone.
But even before we could sequence DNA in entire genomes,
you could do that type of experiment
by using a technique called hybridization,
which I'll come back to.
OK, so the question in this chromosome walk then becomes,
how do you know when to stop?
Because you could do this for a very long time,
but it might not be useful.
So you have to know when to stop,
and you need to know when you arrive at the gene
that you're interested in, which would be the gene that
is responsible for the disease.
So another way to phrase this question is,
how do you know when you have an interesting gene on one
of these fragments?
So let's say this is an interesting gene here.
How do you identify interesting genes?
So now, let's talk about identifying interesting genes.
Anyone have an idea for how they would--
what criteria they would use to define a gene as being
interesting here?
I mean, one could say that all genes are interesting.
If it's a gene, it's interesting, right?
How might we define whether or not there's a gene even there?
It could be-- there could be a gene--
how would you define a gene?
Can someone define for me a gene?
Yeah, Miles?
Is it Miles?
No?
Malik, OK.
AUDIENCE: [INAUDIBLE] that would create a starting and stopping
point.
So like [INAUDIBLE]
ADAM MARTIN: So you'd look for a piece of DNA that
has a start and a stop codon?
So you'd look for an open reading frame, basically.
Yeah.
You could look for an open reading frame.
And so I totally agree with Malik there.
And another criteria you could use
is if it's encoding a protein, at some point,
it also must have been transcribed as an mRNA.
And there are some genes that are transcribed as RNA
but don't make a protein, and they're often
involved in coding or in regulation of gene expression.
So I'm going to--
I'm going to say, is it transcribed?
So is there some transcript that's made?
And specifically, is it transcribed in the tissue
that we're interested in?
So if we're talking aniridia, we might
be looking for genes that are being expressed or transcribed
specifically in eyes.
You're looking for something that
might be expressed in the eye.
If it's not expressed in the eye,
that gene's going to be much less interesting to you
because the phenotype of aniridia is clearly in the eye.
OK, what might be some other criteria here?
Well, one criteria might be, is there a conserved gene that
has an interesting function that's
maybe similar to the disease related phenotype?
So is there a conserved gene with an interesting function?
And to take this example of aniridia,
let's say you're doing this chromosome walk,
and you identify a gene, maybe you
sequence part of this clone, you get a string of sequence,
and you realize that the sequence that you get
is related to a gene from a model organism,
and maybe that gene is called eyeless.
If you've identified a region of sequence in a human,
in the human genome that's mapping to an eye disease gene,
and you find out that in that region,
there is a conserved gene called eyeless,
might be a very interesting gene for you.
So eyeless is a gene.
So here's a normal fly.
You see it has that bright red eye.
The eyeless gene, when mutated, results
in a fly that now just doesn't have a white eye,
but has no eye altogether.
So it turns out that the aniridia gene is the homolog
of the eyeless gene in flies.
That's not how it was identified initially,
but nowadays, there's a lot of information in model organisms.
And so if you're sort of trying to identify a gene,
and you see that there's a gene in the neighborhood
you're looking at with a function that's
related to a gene like eyeless, which
has a clear sort of analogy in terms of phenotypes,
then that's going to increase your interest in that gene.
So I'm going to come back to this point
here, which is how do we determine
whether a piece of DNA that's on one of these inserts
that we're getting as we walk across the chromosome,
how do we know whether it is transcribed or not?
And to get at this, I'm going to introduce you
to a concept which is important in
and of itself, which is the idea of cDNA.
So cDNA.
And specifically, I'm going to show you
how one would make a cDNA library, which is basically
a library of different cDNAs.
And so what cDNA is, as shown up there on my slide,
a cDNA is complementary DNA.
It's complementary DNA, meaning that is the complement
of an mRNA transcript.
This DNA is the complement of an RNA or mRNA transcript.
One thing to watch out for is it's not complimentary DNA.
So this is MIT.
This is a no compliment zone, so I don't want
to see any complimentary DNA.
All right, so let's think about complementary DNA.
So remember, we've talked about the central dogma
and how DNA encodes for RNA, which encodes for protein.
And so the information flows from DNA
through RNA to protein.
But there are some specialized cases in biology
where this information flow is reversed.
So there can be a reverse of information flow
where information flows from RNA to DNA.
OK, so that's pretty cool.
Where does that happen?
Well, there are viruses, such as retroviruses,
one example of a retrovirus is HIV, and the virus life--
the virus genome is a single-stranded RNA molecule,
and the life cycle of the virus is that inserts into the host--
the host genome, which is double-stranded DNA.
For a retrovirus to do that, it needs to take its RNA genome
and make double-stranded DNA in order for it to insert.
So this is an example in biology, which is basically
breaking the rules that we talked to you about earlier
in the semester.
Also, there are retrotransposons which
do a similar process, going from an RNA molecule
to double-stranded DNA.
So this is a specialized case, and it's interesting,
and we can take advantage of it to basically clone and identify
mRNA transcripts.
OK, so I'm going to tell you how to make complementary DNA,
and I'll go through a series of steps.
The first step is we want to make complementary DNA of mRNA,
so we need a way to purify the mRNA.
So anyone have any idea how to purify mRNA?
First, we could maybe draw an RNA molecule here.
What are some salient features of mature mRNA?
Yeah, Carlos?
AUDIENCE: It'll have the five-prime cap
[INAUDIBLE] phosphate.
ADAM MARTIN: Yeah, it'll have a five-prime cap.
Anything else?
Jeremy?
AUDIENCE: Poly-A tail.
ADAM MARTIN: It'll have a five-prime cap
and a poly-A tail.
I'm going to take advantage mostly of the poly-A tail here.
So here, we have a poly-A tail.
OK, how might we use that poly-A tail to purify mRNA?
Natalie?
AUDIENCE: Well, you can add a [INAUDIBLE]
because you know they're [INAUDIBLE]
ADAM MARTIN: Mm-hmm.
What sequence would you use?
AUDIENCE: [INAUDIBLE]
ADAM MARTIN: Yes.
So Natalie has suggested using poly T, which
she said would stick to this poly A tail because
of base pair hybridization, OK?
So let's say we have a bead or some type of resin with dTs
hanging off of it.
So I'll draw a few of them, but you'd
have maybe a lot of them sticking off, OK?
So you have a bead with pieces of DNA, all of which
are poly dT hanging off of it.
And then these poly dTs, if you add cytoplasm from cells,
the mRNA in that cytoplasm is going to stick to this poly dT
bead, and it will stick with a higher
affinity than other things that are non specifically sticking
to the beads, and you can wash these beads with buffer
and salt to get rid of everything that's
non-specifically sticking to the bead,
and then you're left with just a bead that's
enriched with mRNA, which is what was specifically
sticking to this, OK?
So you could purify--
you're purifying the mRNA based on its affinity for a poly dT,
OK?
So then you're going to have enrichment
of mRNA in your sample.
And so then once you have your RNA,
you're going to want to somehow go from RNA to DNA, OK?
So the next step will involve somehow going from RNA to DNA.
So let's draw our piece of RNA here.
Here's our RNA.
It has a poly A tail so it's mRNA.
There is 5 prime.
OK, so now we need to take advantage of a trick.
We can still take advantage of dT
because we can use this as a primer
because polymerase usually requires
some primer and a three prime hydroxyl in order to extend.
Now, can we use DNA polymerase to extend this primer?
Jeremy is shaking his head no.
Why?
AUDIENCE: Because DNA [INAUDIBLE]
ADAM MARTIN: Exactly.
So what Jeremy is saying is DNA polymerase
is a DNA dependent DNA polymerase, OK?
DNA polymerase can only use this if this is DNA here, OK?
So we need a different type of enzyme, essentially,
in order to make DNA from RNA, and luckily,
molecular biologists--
actually one of whom was here at MIT--
discovered this type of enzyme, and it's
called reverse transcriptase.
Reverse transcriptase.
This is an enzyme that's encoded by retroviruses in order
to make double stranded DNA from RNA,
and that allows the retrovirus to insert into the host genome,
OK?
And what reverse transcriptase is is it's an RNA dependent DNA
polymerase, OK?
So it takes RNA as its substrate,
and then it synthesizes DNA on the opposite strand, OK?
So this is an RNA dependent DNA polymerase.
OK, so if you add reverse transcriptase
to mRNAs that have these dT primers, then what you get
is a new strand, which is DNA here.
This is the strand of DNA.
And then you have a strand of RNA opposite it, OK?
So at this step, you have a DNA RNA hybrid.
So this is a DNA RNA hybrid.
Let's see.
Reveal some more of this.
This is the process which I'm basically
outlining on the board.
So then you want double stranded DNA,
so you don't want this strand of RNA that's down here,
so you have to get rid of it.
So you would degrade the RNA, and this
is done using another enzymatic activity, which
is derived from reverse transcriptase, which
is termed RNAs H activity.
So you can add an enzyme RNAs H, which
RNAs H takes this DNA RNA hybrids
and degrades the RNA part of it, OK?
So this is going to degrade the RNA strand.
And if you degrade the RNA strand,
then you're left with a single strand of DNA.
So you have single strand of DNA here,
and now what you need to do is to synthesize
the second strand of DNA.
So you need a second strand synthesis.
And so you need, again, a primer in order
to prime the synthesis here.
So there are a variety of ways to do this.
You can add some type of hairpin,
which is five prime here and three prime here,
and then you can use either DNA, polymerase,
or reverse transcriptase, which also can be a DNA dependent DNA
polymerase to transcribe this strand here, OK?
So again, you add polymerase, and now you've gone
and you've generated double stranded DNA, OK?
So everyone see how we've gone from an mRNA transcript,
and we've done the reverse of everything we just told you
in the first half of the course because we've gone from RNA
and we've made DNA, OK?
But this will be really useful because now we
have a stable piece of DNA that we can clone into a plasmid
and we have a record of this transcript being
present in our sample, and we can propagate that on and on,
so we've cloned it, OK?
All right, what's going to be special about this piece of DNA
versus a piece of genomic DNA?
Natalie?
AUDIENCE: [INAUDIBLE]
ADAM MARTIN: Yes, so Natalie suggesting
that it doesn't have introns, and that's totally right.
So this is not like genomic DNA, and what
Natalie said is because mRNA is processed,
the introns are spliced out, such the mature mRNA only
has the axons, and so this piece of complementary cDNA
is going to have no introns.
How else is it different?
Yeah, Jeremy?
AUDIENCE: It's not going to have promoters.
ADAM MARTIN: It's not going to have a promoter.
Yes, Carmen?
AUDIENCE: It doesn't have [INAUDIBLE]
ADAM MARTIN: You might see a poly A and T
sequence in the cDNA.
Yes, that's true.
OK, so you might have poly A, poly T. I'm
going to focus on the other part from--
there's going to be no promoter, enhancer, regulatory sequences.
Basically, it's got no sequence that's not transcribed, right?
The DNA is only going to have the part of the gene that
was physically transcribed by the RNA polymerase originally.
OK, so no non-transcribed regions.
No non-transcribed regions, and Carmen's absolutely right.
You will also have possibly a poly A or poly T sequence.
OK, so when you get these cDNAs, you might have--
you have more than one mRNA in a sample
like a cytoplasmic extract, so you're going to prime--
you're going to make multiple cDNA
and different cDNAs will reflect different transcripts that
are present in your sample, OK?
So you could have one clone that's
one gene, another clone that's a different gene,
and another clone that's another gene,
and you could have thousands of clones of these different DNAs.
What's going to be special about what types of genes
are you going to get for I guess different tissues.
Are they going to be the same or not?
Yeah, Carlos?
AUDIENCE: [INAUDIBLE]
ADAM MARTIN: Exactly.
You're not going to see-- if you've prepared a tissue
and there is no gene being--
if one gene was not expressed or transcribed in that tissue,
you will not get a cDNA for that particular gene
in your library, OK?
So the representation of genes--
the representation of genes in a cDNA library
is totally dependent on what genes are being expressed, OK?
So this representation is going to be
proportional to the expression level, and the more genes--
the more a gene is expressed in a given tissue,
the more copies of cDNA for that gene
you would see in the library, OK?
So there's really a proportionality
between the number of clones in a library
and the expression level of a gene,
where in the most extreme case, if this gene is not
expressed at all, you're not going
to see it represented at all in the cDNA library, OK?
And then a corollary to this statement
is that if you make cDNA libraries from different cell
types or different tissue types, the cDNA libraries
are going to be different between those different types
of sources of mRNA, OK?
So in other words, different tissues
give you different cDNA.
OK, so there is the process.
So I went through most of the side.
Yes, miles?
AUDIENCE: Is this a way you can determine what gene sequences
are expressed in all cells?
Because in certain mRNA strands across all tissue samples,
those are basic cell functions and expressed in a [INAUDIBLE]
organism?
ADAM MARTIN: So you're asking, if you grind up
like an entire organism and if you
get a cDNA from that library, could you
tell if it's expressed in all different cell types?
Even if you have one cell type that expresses a gene,
if you grind up the entire organism,
then you're going to have some mRNA that represents that gene.
So I don't think it would be as an effective measure
to determine the ubiquity of expression of a given gene,
but in just a minute, I'm going to give you
a tool that would allow you to answer the exact question
that you're asking, OK?
Any other questions about the cDNA library?
OK.
So I just wanted to mention that a comeback to this example I
gave on the identification of the human CDK gene.
So remember, we started with yeast that were mutant.
They had temperature-sensitive mutants,
and we transformed these mutants with a library,
but I didn't really tell you what the library was.
It was in fact the cDNA library from humans that
was transformed into yeast, OK?
And that's because yeast genes--
for the most part, they don't have a lot of interests, and so
the yeast--
the machinery is not able to splice out
the human interactions and human genes, OK?
And so this was done with a human cDNA
library, which then encoded--
one of which encoded the cumin CDK gene, and that
allowed Paul Nurse to discover the piece of DNA that
encoded for the human CDK, OK?
So I just wanted to kind of retroactively
go back and sort of tell you how that experiment was done.
OK, so now I'm going to get to my final point
for this lecture, which is this final technique, which
will allow us to determine whether or not a transcript is
expressed in a single cell type or ubiquitously
through an organism, and this involves a technique, which
is known as hybridization.
And what hybridization is is if you're
starting with a piece of DNA, you
don't need to know its sequence in order
to determine whether there are sequences that
are similar or identical to it, because hybridisation
is basically if you have some sequence
and it's single stranded such that you have a DNA
backbone but you have base pairs that
are able to pair with their complementary bases
and you can use a piece of single stranded DNA like this
and you can label it such that if the labeled piece sticks
to another piece that has identical or similar sequence,
you'll be able to visualize it in some way, OK?
So this is called--
you're looking for things that anneal or hybridize
to a particular specific sequence.
So you don't need to know the sequence a priori, OK?
You just need to have this physical piece of DNA,
and you can use this single stranded piece of DNA
to then fish for similar sequences, OK?
So we could take a piece of DNA here maybe that's in a gene,
and we could fish through a DNA library
to try to identify a cDNA clone that has sequence identity
to that piece of DNA, OK?
And the way this is done is to take a cDNA library.
So each of these colonies here would express or have
a different clone of DNA.
You can then take a nitrocellulose filter, put it
on this plate, which would stick the bacteria in place
to that filter, and you could then lice the bacteria
and denature the DNA, and then the DNA is stuck to the figure,
but now it's single stranded.
You can then add your probe, which is labeled,
and look for the colonies that this probe sticks to,
and that would then identify a particular cDNA, which
would identify whether or not a piece of DNA
is expressed in a given tissue type, OK?
So everyone see how that would work?
So in addition to doing this on a nitrous cellulose filter,
you can also do this in a tissue,
and that's known as in situ hybridization.
And in this case, in situ hybridization,
you're searching for mRNA in a section of fixed tissue.
OK, and I have an example from this paper
here, which is the paper this are cloned.
In this paper was the cloning of the aniridia gene,
and they identified a gene of interest, which is called Pax6
now, and they basically used a piece of DNA
that they thought was interesting,
and they did in situ hybridization in an organism,
in this case, you see an eye.
This is an eye here, and the label Pax6
is labeled in yellow, and you can
see how this transcript is present throughout
the entire eye, right?
And the way you would see if it's tissue specific is you
look in other tissues and you wouldn't see this yellow label.
So that's how you would determine
if it's expressed in a specific tissue or ubiquitously
throughout an organism.
OK, so this Pax6 gene.
Oop.
So I was going to ask, what do you think
would happen if you hyperactivate Pax6 in humans,
and this is one idea, but actually, I just made that up,
or Stan Lee made that up, but actually, Stan Lee never
in fact mentioned whether or not cyclops is a Pax6 mutant,
but we can do a different type of experiment, which might be
more ethical, which is we know there's a fly gene that's
homologous to Pax6.
And what we can do in flies is we can topically
express this islets gene in non-eye tissues
and see what happens.
OK so, this is pretty wild.
This is my Halloween image of the class.
So this is a fly where eyeless has been
expressed all over its body.
OK, so here you see there's an eye--
It's normal eye-- here.
You can see there's now another eye
growing in the front of its head.
You can see here's an eye growing on this fly's back,
and you can see the legs.
There's eye tissue all over the legs of this fly, OK?
So this Pax6 gene, which is conserved from flies to humans
is the master regulator of eye development, OK?
And at least in flies, if you topically
express this in other parts of the body, you get an eye.
I should say these are not functionalized.
They don't hook up to the brain the same way
the normal eye does.
So it's not like this fly can see out
of the back of its head.
OK, that's it.
I'm done, and good luck on your exam on Wednesday.
We will see you here.