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Lecture 5

Lecture 5- Transposable Elements

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University of Toronto St. George
Human Biology
Michelle French

Thursday, February 5, 2009 - Todays lecture is about stuff that should be unfamiliar to us, not really new in terms of science but things that we probably havent learned before and we will get some background on it. - The to p ic is transp sable 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 theyre used in a laboratory setting. - These part icular 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 didnt believe that it was foundin any other organism. Most thought that it was a unique thing to corn so while her results were important, they didnt have a very broad recognition until many years later. It wasnt 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 transp o sable elements that un d erg o transp si toi n are these smal l 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 well 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 didnt 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 e r lo oin g at cel sl wi t the chromosomes so two co p ies 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 colo urles s so the cel ls that have this DN A wi l lbe 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 pigment. - If peo ple fig ured out that yo u 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 youd have these spotted kernels. But you need the Ac present, if you dont 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 www.notesolution.comcause 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 cant 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 abou t 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. - T
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