BiologyFilter: Can I pack chromatin in a test tube?
February 6, 2008 11:02 AM Subscribe
I've finally decided to learn about how DNA (by reading Molecular Biology Made Fun And Simple), and I have a question about chromatin. The pictures you see of chromosomes are all taken when chromosomes have packed themselves into a visible structure to allow them to move around during Metaphase. If I have stranded, intact chromosomes in a test tube, can I force them to pack themselves into the Metaphase configuration? If so, how?
If all you had was the DNA, nope. DNA packs into that structure through complex interactions with a variety of different proteins. Histone proteins in particular, are important, because they're like beads that the DNA winds around.
Here's a picture that helps illustrate histone-DNA interaction.
posted by chrisamiller at 11:34 AM on February 6, 2008
Here's a picture that helps illustrate histone-DNA interaction.
posted by chrisamiller at 11:34 AM on February 6, 2008
Best answer: This depends a little bit on what you mean by metaphase configuration. I would distinguish between a couple different things going on there. First, you have chromosome condensation (going from lots of floppy DNA to tightly coiled DNA). Second, you have sister chromatid pairing (giving you those nice X-shaped chromosomes). Third you have alignment of the chromosomes at the metaphase plate.
A lot of work has been done on reconstituting parts of the cell cycle in extract systems. You can certainly recapitulate chromosome condensation in extracts - see for instance this paper.
You can make pretty decent spindles in Xenopus extracts, but they don't seem to make really pretty paired chromosomes and a nice metaphase plate the way cells do - see figure 1 of this News and Views.
Also, what's in the test tube in the case of these extract systems is a really complicated mixture of stuff made by grinding up whole cells - in the Xenopus case, frog eggs. So it's not as simple as taking a tube with some DNA and throwing in a cocktail of proteins, though most of these researchers are trying to identify what that cocktail would be. But it will probably be a long list of proteins when they find it out.
posted by pombe at 11:34 AM on February 6, 2008
A lot of work has been done on reconstituting parts of the cell cycle in extract systems. You can certainly recapitulate chromosome condensation in extracts - see for instance this paper.
You can make pretty decent spindles in Xenopus extracts, but they don't seem to make really pretty paired chromosomes and a nice metaphase plate the way cells do - see figure 1 of this News and Views.
Also, what's in the test tube in the case of these extract systems is a really complicated mixture of stuff made by grinding up whole cells - in the Xenopus case, frog eggs. So it's not as simple as taking a tube with some DNA and throwing in a cocktail of proteins, though most of these researchers are trying to identify what that cocktail would be. But it will probably be a long list of proteins when they find it out.
posted by pombe at 11:34 AM on February 6, 2008
Oh, misunderstood the question. You really want to replicate the prophase step, not metaphase. The condensation is done by big protein complexes. You might be able to isolate those & throw them in, but I kind of doubt it.
posted by devilsbrigade at 11:36 AM on February 6, 2008
posted by devilsbrigade at 11:36 AM on February 6, 2008
Best answer: Kind of. One system for doing this type of thing is to take the chromatin from sperm, and add it to a an extract taken from a mitotic egg (this is done with eggs from the frog Xenopus Laevis). People studying chromosome condensation use this system, adding and removing components to the egg extract to see what's required for the process to occur.
So adding extracts from mitotic eggs to your isolated chromosomes should do the trick. (I am assuming that by "chromosomes" you don't mean naked DNA, but rather DNA plus histones etc).
Googling with terms such as Chromosome assembly mitotic extract will find you a lot of very technical information, most of which you won't be allowed to access unless you're on a university network or similar.
But for example, here's a recipe.
Disclaimer: I'm a biologist, but this is a subject I know very little about.
posted by nowonmai at 12:54 PM on February 6, 2008
So adding extracts from mitotic eggs to your isolated chromosomes should do the trick. (I am assuming that by "chromosomes" you don't mean naked DNA, but rather DNA plus histones etc).
Googling with terms such as Chromosome assembly mitotic extract will find you a lot of very technical information, most of which you won't be allowed to access unless you're on a university network or similar.
But for example, here's a recipe.
Disclaimer: I'm a biologist, but this is a subject I know very little about.
posted by nowonmai at 12:54 PM on February 6, 2008
nowonmai has nailed it. The technique appears to have been developed in the 80s and has been used ever since:
Newport J, Spann T. (1987) Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly and chromosome condensation are independent processes. Cell 48: 219– 30.
We describe a stable cell-free mitotic extract derived from Xenopus eggs that contains activities necessary for nuclear envelope breakdown and chromosome condensation during mitosis. Using these cell-free extracts, we have demonstrated that nuclear envelope vesicularization, lamina solubilization, and chromosome condensation are independent and separable biochemical processes. We present evidence indicating that during mitosis nuclear membrane breakdown may involve the binding of a coating protein, lamin solubilization is enzymatically driven, and chromosome condensation involves both binding proteins and enzymatic activities including topoisomerase II. These results provide a coherent framework for investigating structural modification of the nucleus during mitosis at the biochemical level.
posted by kisch mokusch at 1:47 PM on February 6, 2008
Newport J, Spann T. (1987) Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly and chromosome condensation are independent processes. Cell 48: 219– 30.
We describe a stable cell-free mitotic extract derived from Xenopus eggs that contains activities necessary for nuclear envelope breakdown and chromosome condensation during mitosis. Using these cell-free extracts, we have demonstrated that nuclear envelope vesicularization, lamina solubilization, and chromosome condensation are independent and separable biochemical processes. We present evidence indicating that during mitosis nuclear membrane breakdown may involve the binding of a coating protein, lamin solubilization is enzymatically driven, and chromosome condensation involves both binding proteins and enzymatic activities including topoisomerase II. These results provide a coherent framework for investigating structural modification of the nucleus during mitosis at the biochemical level.
posted by kisch mokusch at 1:47 PM on February 6, 2008
This thread is closed to new comments.
posted by devilsbrigade at 11:32 AM on February 6, 2008