Thursday, February 5, 2009
- Today’s lecture is about stuff that should be unfamiliar to us, not really new
in terms of science but things that we probably haven’t learned before and we
will get some background on it.
- The topic is transposable elements, which was first identified in corn
(maize). She will talk about how they work, two types that we have in us and
what the effects are in terms of genetics and the last part will be how they’re
used in a laboratory setting.
- These particular elements were first identified in corn and you can see
examples of these corn kernels which have a very unusual appearance, they
have yellow but then they have these purple dots.
- There was a researcher Barbara McClintock who started to work in the 20s
and 30s on corn specifically and she used microscopes to look at what was
going on with chromosomes during the process of meiosis and other times.
She was the first person to identify the crossing over of chromosomes in
meiosis so that was the first big discovery she made.
- The other big discovery that she made was these transposable elements and
she figured these out by looking at chromosomes of different plants, doing
different kinds of crosses and looking to see what the kernels looked like in
terms of colours and she worked out this idea.
- People believed what she had shown but didn’t believe that it was found in
any other organism. Most thought that it was a unique thing to corn so while
her results were important, they didn’t have a very broad recognition until
many years later. It wasn’t until 1983 that she was eventually awarded the
Nobel prize for the transposable element b/c by that time, people realized that
these transposable elements were found in many organisms including us &
there were important implications about them.
- These transposable elements that undergo transposition are these small
segments of DNA that can move from one position of the genome to another.
- We will see the original observations of Barbara McClintock and then we’ll
see how they are more broadly applied.
- So one thing that she noticed while looking at chromosomes of the corn was
that on chromosome 9, the corn has 10 chromosomes, she noticed that during
the paring of chromosomes during meiosis, there was a situation where there
was one chromosome that appeared to be broken, that appeared to happen
frequently and it also appeared to happen around the same place. This part of
the chromosome was lost in the cell or if it was present, was only a little blob.
- Through doing different kinds of analysis, it was found that this region had a
gene called Ds so it stood for dissociator and it seemed to be a place where
you got a break all the time or often. This didn’t happen all the time unless
there was a gene present called Ac so you needed to have Ac present to be
able to see this break in the chromosome.
- When she did different analysis of different corn with different phenotypes in
corn kernel, she saw two types of observations. The first type is shown in a)
and there is a second type down a bit.
- We’re looking at cells with the chromosomes so two copies of the
chromosome you would expect to see. On these particular chromosomes, you
see 2 different alleles, one which has a big C and one with a small c. The big C
in this example means wild type and the small c means recessive or the
nonfunctional. The C gene happens to code for colour so C determines a
purple colour, if you have a big C then you have a purple colour in the cell and
for Sh, the wild type, the big S with the h is shrunken so if you have a big Sh
then it is nice and plump like a regular corn you see, so that is Sh so that is a
nice plump kernel and the Wx is for shiny. If you have the wild type then you
have a purple colour that is nice and plump and shiny in appearance.
- As the kernel grows, in some of the cells, what happens is, because in this
region the Ds region, if it was present (located in the slide), as the kernel
grows, some of the cells get a break at the Ds so wherever that Ds is located
you lose that part of the chromosome. That part becomes nonfunctional and
gets destroyed. Cells with this particular genotype, they take on a different
phenotype – they are colourless so the cells that have this DNA will be
colourless, shrunken and not shiny (dull).
- This would be happening during the development of the kernel so you have a
region that is colourless, shrunken and not shiny and then you have the normal
pigmented plump and shiny.
- In other cases, there was another type of allele at the C locus so in other
words, at the position where the colour gene was, that was a different allele
(different sequence). In this case, the cells would inherit a different
combination of genes & alleles so you would have dominant ones & you have
the recessive ones. The deal with this c-Ds is that the Ds is inserted into that C
gene, the big C gene and therefore notice how it has a small c there, it means it
would be colorless so not normally allowing the purple colour to show. The
reason is because the DS has been inserted into the normal C gene. What
happens is the cells initially have this genotype and as they develop, some of
the cells have a situation where the Ds actually moves out of that region where
the C gene is and when that Ds chunk moves away, you now return to the
normal wild type gene. Therefore what you end up with are regions where the
cell has gone back to the regular phenotype with purple & spots.
- This is what Barbara McClintock figured out, that there were parts of the
DNA that were moving around, that this Ds had the ability to move out of a
region of a chromosome and therefore change the phenotype.
- Over time with experiments, people realized that the C gene would look like
this, and it would code for purple pigment. Or if you had a situation where you
had a copy of this, you would have a purple pigment.
- In the case of a mutant with a cm mutant, or the c-Ds mutant shown on the
other slide. In this case the C gene is interrupted by this Ds element it is called
a transposable element. This interrupts the coding sequence of the gene &
therefore in this case providing this stays sable (?), you get a colourless
- If people figured out that you had the Ac gene which is a completely
different gene and it was present with the cm-DS gene, so the same one as up
there then in certain circumstances in certain cells, the Ds could move out so it
would move out of this locus where the C gene was and then you’d have these
spotted kernels. But you need the Ac present, if you don’t have the Ac present
then you always have colourless and as well there was another mutant that had
Ac present in the same region in the C gene and Ac on its own was able to
cause its own movement. So Ac could move and could move itself out of the
region so you would get spotted kernels again.
- What this tells you is that with these spotted kernels is that you see a
situation where the DNA is literally jumping from one place, it is jumping out
of the chromosome. This is something people thought was impossible, that
DNA could move around like this.
- The DS is called a non-autonomous element because it requires the Ac so it
can’t work on its own. When it is autonomous you can do what you want. In
this case it is not autonomous, it can move but you need Ac whereas Ac is
called an autonomous element because it can move itself around the genome.
- Now for the mechanism about how the phenotypes appear. We delve into
what the Ac is and how the Ds actually moves.
- Here is the situation: the Ds and the Ac are both transposable elements and
the Ac actually codes for an enzyme that is called transposase. The Ac is
coding for an enzyme called transposase. What the transposase does is it binds
to the specific sequences that the ends of the transposable elements have, so
notice here it is shown called inverted repeats, the purple parts at the ends of
the Ac. The elements have these sequences that the transposase recognizes.
The transposase binds to those regions and causes a twirling around, forming a
loop in the structure in the center. What happens is the transposase cuts the
part out that is between the inverted repeats. It can go and recognize other
sequences in the genome and insert that piece into those places. It can actually
insert the DNA into another location. In this example, it is cutting out the
transposable element and moving it to a new location.
- These would be the sequences in the example where the DNA gets cut.
These are the flanking regions of the DNA of the transposable elements. This
is the sequences recognized by the transposases. The transposase recognize
this specific sequence, cuts it and inserts in a transposable element that may
have come from somewhere else and the host will repair the gaps.
- You notice the sequence remains once it has been cut so you have these
flanking repeat regions that will be present once again.
- That is how it works so that gives you more detail, the Ac codes for the
transposase and the Ac or the Ds have these flanking regions that allow the
DNA segment to move around.
- That example shown on the previous slide with Ds is an example of a
conservative method of movement.
- In the case for example this is a particular transposon called TN10 and what
happens in its case is it will get cut out of one region, shown in the slide
between A and B. A and B will get rejoined together and this TN10 will move
to another location so that is called conservative transposition – there is no
increase in the number of transposable elements, it is just being moved from
one place to another.
- The other type is called replicative and we will see how that works in more
detail but notice in the replicative we start with the two products. These two
regions could be on the chromosome or different chromosome and this TN3 is
going to get copied and then the copy will be inserted into another region. You
keep the original where it was. In this case, this is called replicative because
notice now, we have two copies of that transposable element whereas
previously, we only had 1.
wanyiwu and 39450 others unlocked
HMB265H1 Full Course Notes