Unformatted text preview: This is called kinetoplast DNA. it's called the kDNA nucleoid body. There's a single ... It's very highly flagellated. Lemme just tell you how these cells divide. The first thing that happens is tha tyou start seeing the kinetoplast divides first so you see two little blue dots. Then the nucleus divides. And then they sort of pull apart. The S phase is fairly synchronized. This is just my diagram they overcome the mitochondria. It circularizes and when they divide, they have the asymmetric mito. This is T./ brucei. Here is a kinetoplast in the late stage. It gets elongated. This is a transverse section. There's a puffing of the fibers heer. See here, you have the two basal bodies and they're probably what pulls the whole mitochodnrion apart. When you isolate the kinetoplast and you add the detergent and burst out the cell. You can centrifuge down the DNA at about 4000 G and the problem is the nuclear DNA is very viscoius. So the moment you lyse the cell, it's everywhere. So we broke it up with a syringe and so that it wouldn't viscious anymore. So then you do a low spin and you get the kDNA. You get things that look liek this stained with DAPI. Crithidia is a parasite of insects only. It's called monogenetic because it doesn't have a vertebrate host. Digenetic is trypanosome and leishmania. All others in this genera are mongenetic. You can grow them in a fermenter and get them in high density per cell. They're very easy to manipulate. They even use transformation with it. In the electron micrograph, there's an edge/. This is just DNA. no protein involved. Then you have these lolong molecules. If you count the minicircles in crithidia, there are about 5000 minicircles. if you treat with toposomerase type 2. Nicks twice and then passes the DNA through and reseals it. I just want to put this network into some kind of evolutionary perspective. This was done with ribosomal RNA. T. brucei and Euclena brance right here and then they branch to the kinetoplastids and the eugenoids. In this region you see two main groups. Crithidia, leishmani,, leptomonas, theere's even a plant parasite. This is why the palm trees are dying in BH. We think there's a single ancestor for all this group. They all come of oone branch. If you look at their kDNA, you see them all in a network. The only one able to be grown other than this group is T. borrell without bacteria. They don't have minicircles networks. It's all disorganized. We think catenization occured in the other ancestors. When we first dicovered we could see the networks in the microscope, we found that we could see them and we could do things with them. We could see where replication of minicriclse. We didn't know what they were. Didn't know what they did. It was mystery for a long time. We labeled with tridium. We used it with autoradiography. It's used for light microscope with tridiated thymidine. It's a DNA precursor, they take it up easily. We gave a one minute pulse and then covered with a liqueid emulsion. Just like the meulsion on film. We called them antipodal nodes because they were always 180 degrees. We saw this thing, the rings were around the periphery, no longer in the two little circle.s Paul Englund. We also found in the early experiments, they were open cirels If it's closed, there's no nicks and they're super twisted. If you have a nick in one or both strands, it's an open circle. If you add ethidium bromide to a open circled, it'll get bigger. If you add it to a closed circled, it will twist up to a small rod. This is a fractionation until you reach the same density. There's two forms, which is most dense. Another appeared called form 2. Form 1 was closed and form 2 is open. Paul repeated what we had done. These were labled with nick translation. It will label at spots where there are nicks. B is covered with emulsion and saw several types. Depending on where they were on that emulsion. After 4 months, he saw a ring that we saw. It shows there are nicked minicircles around the edge. Now we know they are opened circles when they replicate. When he looked at different places, D is form 2. It was totally labeled. The whole network was made of nicked circles. That is strange. Before I get to that, he also looked at it morphologically, he spread them on ethidium bromide. If it's super twisted, he can see it. The other ones are open molecules. It confirms what saw in that trnaslation. There's open circles outside. C is later on, in the cell cycle. Now he ses closed circles just in the center and all these are nicked. Again confirming nick translation. Somehow, there are newly replicated minicircles are nicked and they remain nicked for the entire Sphase. They get these antipodal nodes and then there are no more nclosed circles and then at the end of S phase, they are all ligated together and they become closed. This is the way Paul explained it. Originally there's a form1 network where they're all closed. These are the replicating ones. They get longer and finally fill up the whole thing. Then they get two repairesd and then we get two form1s. I couldn't figure out. Somehow these minicrcles must go through this. Paul figured it out. He stained with DAPI. Here are minicircle probes. It's labeled with fluorescent dye called FITC. In form 1, youc an't hybridize. When he goes to the next one, he seese the two antipiodal nodes and then het gets this donut and then you get these totally fomed tube and then they become form 1 and divide. Thos were done by isolating networks. This was done in vivo. he took log phase crithidia and used RNA probes and he saw two dots. They turn out to be the two nodes. But this is invivo. They probably correspond to that blob where the sprials start to beomce blown up. He looked for DNA polymerase. These are intact cells now. He sees two dots. The DNA polymerase is localized at the two nodes. He said, it wstarts out with Form1. There is random selection of minicircles by topomerase. Then it moves to one or the other antipodal node. This is shown in red. They turn out to be really important. He thinks these are complexes of replication enzyme. Now, how do we explain it becomes the donut. We got this prolifeariting label. Then we did a chase. So we washed and then pulsed. We can see where it groes. The whole ring migrates to the center. At the very end, it goes into the center an dthen these things divide. The next cell cycle is all random. It's trhought the whole structure. Paul did the same thing in the EM. Here's a 3 minute pulse. Notice there's a gradient in B. This just shows again what he sawa gain in more clairty. he got these two p...
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- Fall '08
- DNA, Ethidium Bromide, Paul Englund, little blue dots, kDNA nucleoid body, antipodal nodes