My name is Sue Wessler. I'm a professor of Genetics at the University of California, Riverside,
and my lab studies what is the subject of the talks today: and those are transposable elements.
The title of the general lecture is The Dynamic Genome and the title
of this first presentation is Introduction to Transposable Elements. Here I will talk about the discovery
of transposable elements, and how this seemingly trivial discovery
led to what is now recognized as a revolution in biology. So I start showing pictures
that are integral to the talk. The first is a picture of Barbara McClintock who
is the discoverer of transposable elements, and I'll tell you more about her as the talk
goes on, and the other is the corn kernels which are also integral to the discovery.
So I've divided the talk into three parts. The first is a description of the discovery
of transposable elements by Barbara McClintock. The second is a little more detailed
on how transposable elements actually move and increase their copy number
in the genome. And the third is just how abundant transposable elements are in genomes,
and this is really the part that made transposable elements more than just a trivial
discovery and that has led to transposable elements being viewed as the major
component of the genomes of higher organisms. Here's a picture of Barbara McClintock
when she was a graduate student. It's a picture of the lab of R.A. Emerson
who is the father of maize genetics. This picture was taken at Cornell
University in 1929 and in the next talk actually we will come back and revisit
this particular area of Cornell. Here's a picture of McClintock and you can see
that she wasn't wearing what women normally wore back then - that's a skirt - she wore knickers.
I had the good fortune to know her later in her life and I will talk about that a little later.
The focus of their lab was corn and corn genetics, and one of the reasons
it was such a wonderful organism to study genetics is it had an abundance
of interesting visible characteristics - traits. As we found out years later,
one of the reasons for this diversity is that it has a remarkably diverse genome. So McClintock
asked a very simple question, and this question is really at the heart of this talk
and the next talk. And that is "why are these corn kernels spotted?" Now many of you may recognize
this corn although it doesn't look like the corn you eat as the corn
that you see during Thanksgiving. It's usually hanging up in supermarkets.
It's Indian corn. It's known as Indian corn and unlike the corn we eat which is yellow, this corn is highly pigmented.
But what's unusual about this particular corn cob here are the spotted
corn kernels. McClintock was interested in what is the genetic mechanism responsible
for this spotted kernel phenotype. Here's a picture. A lot of McClintock's
work because she went on to be world famous is available online and you can actually
access her notebooks. This is a picture of her notebook with her typing
at the bottom describing these kernels and the genetic basis for their unusual phenotypes.
The one thing you'll notice is that the kernels are incredibly detailed.
That is one of the reasons that she was able to figure out so much about the behavior
of the transposable elements that I'll talk to you about because of the wonderful resolution
of the phenotypes in the kernels. I'm going to cut to the chase
and tell you McClintock's solution to the spotted corn kernel question and that is that
she discovered that spotted corn kernels were caused by a new type of genetic
element - a new mutation - and I've diagrammed it here. If we start with the gene,
the gene is mutant but not due to a basepair change or deletion, it's due to actually an insertion
of a piece of DNA into the gene and this insertion inactivates
the gene. But unlike other mutations such as, as I said before, basepair changes or deletions this mutation
is reversible. The way it's reversible is that the piece of DNA - the TE or transposable
element and you'll see this abbreviation throughout "TE". The TE can insert into the gene
and it can then excise from the gene. When it inserts we have a mutant phenotype.
When it excises we revert to a wildtype phenotype or normal phenotype.
What I've drawn here is a spotted corn kernel.
And I've tried to explain how the different sectors in the kernel arise.
What you see at the top is a mutant
gene and so we can imagine that this gene is a gene responsible for pigment biosynthesis
so that if the gene is mutant there's no pigment - the kernel is yellow.
If the gene is wildtype, normal, the kernel is purple. To explain a spotted kernel
we have to have a reversible phenotype, a reversible mechanism.
So what you see is that the colorless areas are due to the gene at the top.
That is cells that have the gene at the top that is a gene that is disrupted by a transposable element
so it cannot produce the products necessary for pigment biosynthesis
and so we end up with the yellow unpigmented areas. However, during kernel development
the transposable element can excise from the gene and when it does that it restores expression of the gene
and we end up with the spotted kernels and so because kernel development is very regular
- that is that unlike in animal systems
plant cells when they divide they don't move around they divide and they're literally cemented in place
- we end up with these sectors. A large sector is due to a cell
in which the transposable element has excised from the gene and all of the mitotic progeny
then can express the gene so we end up with a sector much like a clone on a Petri dish of bacteria.
As I said before, the rest of the kernel are from cells
that still have the transposable element in the gene.
McClintock was able to use the behavior of transposable elements and the detailed resolution
that the corn kernels facilitated to understand something about the behavior of the element
and that's shown in this slide.
What we have here at the top is a gene that's pigmented
and it's pigmented because the color gene is wildtype.
That's one allele: a wildtype gene. We have another allele where we have what I call an 'NTE'.
It stands for non-autonomous TE and I'll explain that in a second.
What you're going to see here is that there's two different types of transposable elements.
One is autonomous, one is non-autonomous and these slides will hopefully clarify what I mean by that.
In this case we have a transposable element sitting in a gene,
but the transposable element, which is non-autonomous, is not able to move on its own.
However, that transposable element can move if there's a second transposable element in the genome
and that's shown here as the 'TE' and this is elsewhere in the genome.
It could be on a different chromosome. It is not near this color gene,
but that transposable element activates the non-autonomous element
and causes it to excise from the gene and we end up with a spotted kernel.
In the absence of that autonomous element as you see above it,
the colorless kernel, the transposable element cannot move.
That's why we call it non-autonomous.
And finally the last situation is we can have autonomous elements inserting into genes and that's shown here.
We have the autonomous element inserting into a gene and because the autonomous element
makes everything that is needed for transposition that kernel will be spotted.
To review: McClintock not only discovered transposable elements
but she also discovered that there are different types of transposable elements
and in this case we have the autonomous element and that's defined as an element
that provides everything needed for transposition
and non-autonomous elements which can only move or transpose in the presence
of the autonomous element that is if the autonomous element is in the genome simultaneously.
McClintock made her discoveries initially in the 1940s - a very long time ago
- but it took a long time for the scientific world to catch up with her.
That's why I say here that she was well ahead of her time. In the 40 or 50 years after their discovery,
transposable elements which were initially only recognized to be present in maize
were found in many organisms in fact in virtually all eukaryotes.
In the 1950s transposable elements were discovered in Drosophila fruit fly.
In the 1960s, they were discovered in bacteria, in E. coli.
And in the 1970s they were discovered in the human genome
as a cause of some mutations in the human genome, and we'll talk a lot about that, more about that later.
So here is one of my favorite pictures.
It is a picture of a transposable element in action in a rose in the Napa Valley.
And another picture from my colleague Tom Gerats is a picture of a petunia flower.
And the reason that I show you this is that if you look in gardens, rose gardens or other gardens,
you will notice these phenotypes.
They are not just patterns, which you see with a lot of flowers.
They are actually sectors and in case these sectors are completely analogous
to the spots on the corn kernels that I showed you before.
So McClintock as I said, it took 40 years really for the world, the scientific world,
to recognize that her discovery in corn, in maize, was true for most eukaryotes.
And in fact what McClintock discovered was that there was more in the genome than just genes.
She discovered a new component of the genome.
And I've drawn that here as a chromosome before McClintock.
showing these rectangular boxes, and those are representations of genes.
So before McClintock, people thought, when they thought about it at all,
that the genome, that the chromosomes, were essentially genes sort of lined up like beads on a string.
After McClintock, it was recognized that there was another component in the genome.
And that component as I have drawn as black ellipses here were the transposable elements.
So McClintock recognized that these transposable elements were moving around were not coming from
the environment. They are not viruses that are infecting the organism.
They actually are residents of the genome, and these we now know are residents of most eukaryotic genomes.
So for this discovery McClintock was awarded the Nobel Prize in Medicine or Physiology in 1983.
Now Nobel Prizes are frequently awarded many years after the discovery.
40 years is a very long time, even for Nobel Prizes.
In this really is for the reason I said before. She was really ahead of her time.
The other thing is that Nobel Prizes are frequently awarded for up to three people.
She was awarded the Nobel Prize by herself,
and this really recognizes that this was her discovery.
So here is a picture of her. She lived from 1902 to 1992.
I had the wonderful fortune of knowing her for the last 10 years of her life,
and continuing on with some of her discoveries, and I will be talking about that in the next talk.
She also commented on how her life really encompassed the entire history of genetics.
So Mendel's laws were rediscovered in 1902, the early part of the 1900s, which was the year of her birth.
And she lived long enough first of all to be recognized for her great discoveries, but second of all,
to actually see the world enter the genomics age.
So the age of DNA sequencing, and that is what we will talk about in a minute.
So what I am going to do now is tell you a little bit about how transposable elements move.
So we are going to move from the genetics to the molecular biology.
So here is a diagram of what a generic transposable element looks like.
And so they are very simple genetic systems.
This is a piece of DNA. It varies from a couple of hundred nucleotides to several thousand basepairs.
Transposable elements are.... this is an autonomous element,
so remember this is the element that can move on its own.
It encodes everything needed to move itself and to move a non-autonomous element.
So when it says everything needed, that is a single protein that it encodes, and that is called transposase.
And I will tell you in a minute a little bit more about what transposase does.
The element is flanked by special sequences which are called terminal inverted repeats.
These are sequences that are the same sequence forward and then flipped over backwards.
And I will show you in a minute why, what the functional significance of that is.
And the whole element is flanked by a target site duplication. A 'TSD'.
And I will show you in a minute how that is derived.
So I mentioned that there are autonomous elements and non-autonomous elements.
Non-autonomous elements which cannot move on its own.
They cannot move on their own.
And they require an autonomous element to provide the stuff needed to move them,
and you can see here that what we have is a non-autonomous element is sometimes,
but not always, a defective version of an autonomous element.
So what I have drawn here is a deletion that has been sustained which prevents the non-autonomous element
from making transposase.
The transposase that is made by the autonomous element, as we will see in a second,
can influence both the movement of the autonomous element and the non-autonomous element.
So that is shown in this slide.
So what I've shown here is the transposase, I am sorry, the autonomous element, which is at the top,
encodes a single protein, and that protein is a transposase. And what that transposase does initially
is it binds to the ends, to the terminal inverted repeats,
of both the autonomous element and the non-autonomous element.
So one of the functions that the transposase has is as a DNA binding protein.
Ok, so this cartoon sort of will take you through the steps in the transposition
of a transposable element.
So I've shown here that this is what we saw in the previous slide, but in a more abbreviated form.
We have the transposase proteins bound to the ends of a transposable element
Those transposase molecules come together and form a dimer.
They then cleave the transposable element out of what is called the donor DNA,
out of the rest of the DNA, and that entire complex as you see here then can insert somewhere else.
So what we end up with is a transposable element in a new location in the genome.
So I want to define another term and that is a transposable element family.
And this we will see a lot more in the next talk.
So a transposable element family, as you saw before, has autonomous elements and non-autonomous elements,
and there can be lots and lots of members of the family.
So we can have one ore more autonomous elements,
one or more... many, many non-autonomous elements in the genome.
And as you see here the structure of the non-autonomous element can vary.
Sometimes it will be a simple deletion of the transposase region.
Sometimes it will be more extensive, and sometimes there may be none of the...
the only thing it may share with the autonomous element
are the terminal inverted repeats and the length of the target site duplication.
So what I am going to do in this slide is to show you how the target site duplication arises.
So what you see at the top is going to be a new insertion site of a transposable element.
So this is a piece of DNA where the transposase is going to bind.
It is going to... we talked before about the transposase binding to the ends of the transposon.
Now that is not... this is different from that. This is the target site.
This is where the transposable element is going to insert.
The transposase has a lot of different functions built into this single protein.
One of the functions is to cleave the target site.
And it cleaves it in a way that is much like restriction endonucleases.
It makes a staggered cut, so you see that here.
It cuts on the two strands of the DNA, at essentially the same sequence, and when those strands come apart
you see that we are left with these overhangs.
These sequence overhangs.
It is into this region that the transposable element inserts.
Then we are left with these gaps on the side. Those gaps are filled in by host enzymes.
And because of this reaction, the transposable element, which is shown in green,
is then flanked by a repeat, a target site duplication,
which is a... and the length of that duplication in this case,
we show that the transposable element is cleaving 5 basepairs. It is a staggered cut of 5 basepairs.
The repeat sequence will be 5 basepairs.
Different transposases have characteristic staggered cuts.
So some transposases will cut three basepairs apart. Some will cut 8 basepairs apart.
And the resulting transposable element will then have a target site duplication
that is the length of the staggered cut.
So again here to remind you, here is our transposable element family.
And this is to show you that the families, like all families, like human families,
can be different, can look different.
What we see here is that there could be multiple autonomous elements in the genome.
There could be many, many, many non-autonomous elements in the genome.
And we will see more in the second talk.
The other thing that differs is that genomes can and do have multiple families of transposable elements.
So I've shown you here at the top the family we've been working with
with where the transposase, the blue family,
where the transposase is produced and binds to all the yellow terminal inverted repeats.
At the same time the genome could also have a different family.
And I've shown the green family at the bottom.
The transposase from this family binds specifically to the purple terminal inverted repeats.
So in this way there can be multiple families that co-exist in a genome
that have really nothing to do with each other.
What I want to do now is in a few slides take you through... What I haven't explained to you
is... I've told you that there are multiple copies of transposable elements in the genome.
But I haven't explained how a transposable element can increase its copy number.
Because in fact what I have told you is sort of the opposite.
What I have told you is that a transposable element at one place
excises and moves someplace else. And I am not really great at math,
but I can figure out that that won't increase the copy number.
You start with one. You move from one site; you move to another.
So I want to show you in the next couple of slides is, just very simply,
I'll show you a schematic on how by doing that a transposable element can actually increase its copy number.
Because the copy number of transposable elements will be a major part of the talk, the rest of this talk
and the talk that follows.
So what I have drawn here is at the top I have a transposable element at a particular site
in the genome. That region is now ... the DNA is being replicated.
And what we see is the familiar replication fork. So here are the sister chromatids. OK,
so what happens, this is the same thing at the top, we are replicating.
The transposable element is going to move from one of the sister chromatids after replication
to another site, to the other sister chromatid.
And that is shown at the bottom here.
So I've redrawn that at the top and what happens after this is kind of neat.
The site, the empty site here, sometimes it remains empty,
sometimes the host will use the transposable element on the sister chromatid
to copy it into that empty site, and that is what you see at the bottom here.
So when these chromosomes... when replication is finished,
and we end up with two double stranded daughter strands,
what we have is the top strand has one transposable element,
and the bottom strand has two transposable elements.
So we've gone from a situation where we had two transposable elements,
I am sorry, one transposable element,
to one that now we have two.
These chromatids will separate and they will go into separate cells.
So one cell will have two transposons, and one will have one.
And I think in the next slide what I have done is I have summarized all of the steps.
So we start out with a single transposable element.
There is replication. There is transposition that occurs from one sister chromatid to the other.
There's repair of the empty site using the transposon from the sister chromatid.
And there is separation of the chromatids, and we end up with an increase in one transposon in one of the cells.
That is one mechanism. There is another mechanism which I have just drawn as a shortcut here.
And that is you see at the top just what we started with before. We have a transposable element.
We have replication. We then have transposition not from a replicated site into another replicated site,
but instead from a replicated site into an unreplicated site ahead of the replication fork.
What happens then is again we will have the completion of replication,
and we end up with a separation of strands,
and we end up again with a gain in the number of transposable elements.
So I have told you so far I have focused on one... what is now known to be one
of two classes of transposable elements that are in the genome.
The elements that were discovered by McClintock and that are responsible for the unstable,
for the spotted kernels, the sectored flowers,
those are caused by the element type that I have shown you here
with a transposase with terminal inverted repeats,
but there in fact is another class of transposable elements that I am not really focusing on in this talk.
And I am only going to mention it briefly.
But these are in fact incredibly abundant transposable elements.
They are called retrotransposons.
And these are called now Class 1 transposons.
Whereas the DNA transposons that McClintock studied are called Class 2 transposons.
Now the retrotransposon is characterized by terminal inverted repeats,
long terminal inverted repeats, where the ends of the element are not inverted repeats like the DNA transposon,
they are direct repeats which are shown here.
They also have a target site duplication because
the insertion occurs in the same way as the insertion of DNA transposons.
And now I am calling them DNA transposons and RNA transposons
and the reason is they're named for the intermediate in transposition.
So a DNA transposon excises from one site as a DNA element and moves elsewhere.
In contrast, an RNA transposon or retrotransposon, the intermediate is RNA,
and I've summarized that in the next slide.
So here I have shown you a retrotransposon sitting inserted into a chromosome somewhere.
The element is transcribed much like a gene.
That RNA then is converted into... it is copied into DNA, into a DNA copy.
And this is copied by an enzyme called reverse transcriptase,
and it is encoded by the retrotransposon.
That is then converted into a double stranded DNA molecule.
And that double stranded DNA molecule inserts elsewhere in the genome.
So this is a lot easier than the DNA transposition mechanism that I showed you before.
In essence you could think of a retrotransposon as like a printing press.
It makes RNAs and each of those RNAs potentially could be converted
into a double stranded DNA, and that double stranded DNA can insert elsewhere in the genome.
So a single element because there can be many, many transcripts that come from a single element,
a single element could potentially lead to hundreds and hundreds of new
integrations, of new copies in the genome.
So I've told you about the discovery of transposable elements.
I've told you about, something about how elements move and increase their copy number
Now I want to sort of wow you with something that I think
has been one of the major findings of this era of genomics,
and that is just how many transposable elements there are in the genomes of higher organisms.
So, when we started sequencing genomes,
and when I say we I am using the general "we" of the scientific community.
It turns out that the largest component of genomes are derived from transposable elements.
50% of the human genome, of the chimp genome, of the mouse genome,
more or less fifty percent are derived from transposable element sequences.
Plants, especially flowering plants have even higher proportions of their genomes that are transposable elements.
The maize genome, over 75% of the genome is derived from transposable elements.
The barley genome, and we will be talking more about these creatures in the next talk.
The barley genome is almost 85% transposable elements.
And even more remarkable, the iris genome is 98% transposable elements.
And I don't know about you, but if I look at an iris plant, I mean they are beautiful,
there is nothing that would tell us that their genomes are just largely transposable elements.
I want to give you a feel for how many transposable elements there are in the human genome.
So our genome is comprised of 2.5 billion basepairs.
Let's call them A, G, C, of T. Let's say that's 2.5 billion letters, like A, B, C, and D.
So if they are letters, let's start filling up some books.
So this is equivalent to about a thousand textbooks of a thousand pages each. No pictures.
Only 20 to 40 of those 1,000 textbooks contain all of the genes necessary
to encode the proteins that make us up.
Five hundred of the 1000 textbooks contain sequences that are derived from transposable elements.
So it is pretty stunning.
To give you and idea of just how many transposable elements there are and where they are
I am showing you an example of a typical human gene.
And what you see are the green boxes which are the exons,
coding regions, and the blue areas which are the introns.
Let's look and see where there are transposable elements in this gene.
Because there are transposable elements in 70-80 percent of our genes contain transposable elements.
So this shows you all the different places that this genes has transposable elements.
What you'll notice, it is kind of hard to tell in this slide, but that mostly the non-coding regions,
the non-exonic regions contain some transposable elements.
In fact some human genes have almost a hundred transposable elements in their introns.
So transposable elements are really everywhere.
So what I want to show you in the next slide is how transposable elements
can diversify a group of very, very closely related organisms.
And for this I am going to go back to plants, and I am going to show you
a group of organisms that we are very familiar with - the cereal grasses.
You may not know I came from New York City,
so I didn’t realize that all of these were in fact members of the grass clade.
They are rice, sorghum, maize, and barley are some of the most important organisms
on this planet for human calories.
And what you see here, we've talked about maize a lot.
Maize... the maize genome size is 2500 megabases.
That's about the same size as the human genome.
However, what you see is that relatively closely related organisms,
such as the rice genome has a genome size that is almost ten times smaller, 350 megabases.
Sorghum is twice the size of that, 700 megabases. Barley is 5000 megabases.
These genomes have gone through a remarkable expansion.
An explosion.
And what's responsible for that largely is the increase in the genome size due to transposable elements.
How do organisms function with that much stuff?
There is actually three major reasons for the success of organisms
despite being crowded with that many transposable elements.
The first thing is that most of the transposable elements in the genome
are dead. And when I say dead, I mean they can't move.
They don't move anymore. They probably haven't moved for a long time.
And they are dead because they are mutated. They contain mutations.
So every single generation mutations are introduced into our DNA.
Mutations that occur in genes will lead to problems for the organism, and they are selected against.
However, mutations that occur in transposable elements just accumulate.
It's not a problem to the organism at all.
So we end up with most of the transposons, the vast majority of the 50% of our genome
that are transposable elements are not able to move around anymore.
And will never move around. They will just sort of, as we say, senesce.
The sequences will mutate and mutate
until there is really no trace that they ever were derived from a transposable element.
The second way that organisms survive, it's really how transposable elements survive,
is that transposable elements have evolved mechanisms that allow them to insert into places in the genome
that won't harm the organism.
So for example, one safe haven might be into another transposable element.
So if a transposable element inserts there, it is not inserting... it is not causing
a mutation in any of the genes necessary for the organism to survive.
And we will talk a lot more about that in the second talk.
The final thing which I am just going to allude to briefly here
is that the host has a way to fight transposable elements, and it is a very sophisticated way.
The host silences transposable elements using epigenetic mechanisms and inactivates them.
And I've... I'll show you on the next slide.
So this is just an example of a region of the barley chromosome.
And what you see are... remember I said that 85% of the barley genome is derived from transposable elements.
And this is how a region of the genome might look.
So here we have a few genes, the little blue boxes over there.
And what you see stacked up here are all the transposable elements.
They are the vast majority of this particular region of the gene.
So, but if you look at a flat version of the DNA,
if you actually look at the three dimensional structure, the chromatin,
the way the genome exists, what you find is that the region where these transposable elements are clustered
in fact is a tightly compacted region of the genome.
It is what's called heterochromatin.
There's very little, nothing really... This is a host response.
It condenses the chromatin and prevents the transposons
from making the transcripts and proteins needed for it to move.
So they are said to be silenced.
This in addition to the fact that these transposable elements are accumulating mutations.
In contrast, the region where the host needs the genes to be expressed,
those are euchromatic regions. They are less condensed.
So the genome, the chromosome, is composed of regions that are very, very densely compacted,
and that is generally where the transposable elements are,
and regions that are much less compacted so that the host can access
those to make the gene products needed for its development in life.
So like many things with transposable elements, McClintock was ahead of her time.
She didn't only discover transposable elements,
but she proposed that they had a role in generating diversity.
And this scenario had the following components.
That transposable elements in the genome usually do not move around,
because if they did move around, they would cause mutations.
That "stress" conditions may activate transposable elements.
Now when I say stress conditions I am thinking more... I am not talking about like driving in rush hour traffic,
I am talking more about climate change, that might be one thing.
This is a scenario. This is not proven by any means.
This was her ideas 20 or 30 years ago.
So genomes have a stash of transposable elements. Most are inactive, some are active.
And the host is keeping them inactive by these epigenetic mechanisms, but if something happens to the host,
some of these, or the host population, some of these transposable elements can be activated to move around.
The significance of that is that the movement of transposable elements will generate genetic diversity.
And it will do this by increasing the frequency of mutation.
So what we end up with is a population that is now more diverse because of the movement of transposable elements.
There is new mutations in that population, and it is possible that some of those new mutations may be adaptive.
May help the population survive this dramatic change in climate, or whatever.
So I want to end by essentially saying to you that the way I think of transposable elements
is that they shake up the genome. And the genome is inherently conservative.
So they shake up an otherwise conservative genome in ways that we are jut beginning to understand
and ways that I will go into in the next talk.