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Komplizierte Wissenschaft in einem passenden Format zum Bier – Der Science sl6
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Tag: Telomere
Transforming Yeast or how to make a GMO
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In the last article I ended with a DNA fragment, that I created with a PCR. Now I need to get this DNA into my yeast cells to teach them to age like normal people. This is called transformation. There are several different transformation techniques but most common are elctroporation and chemical transformation. You can spark emotional debates if you ask what is better but the more common and generally a bit easier is the chemical transformation. That is also the method I chose for my aging yeast.
How transformation works mechanistically is not completely understood but the main idea is that you have to overcome the barriers protecting cells from their surroundings. These barriers are the cell wall (for all except animal cells who don’t have one) and the cell membrane. The cell wall’s main responsibility is mechanical stability. You can imagine it like a steel frame that prevents the membrane, which you can imagine like a water bomb, from swelling too much until it bursts. On a molecular level it is a relatively wide net, so it’s not a physical barrier for the DNA molecules. What’s problematic however is that it is electrically charged. DNA is negatively charged and would be repelled from the mostly also negatively charged molecules in the cell wall. This obstacle is overcome by adding positively charged ions that neutralize the charges of the cell wall and allow the DNA to get close to the cell’s membrane.
But than it has another problem which is the cell membrane. The membrane is a double layer of lipids, fat molecules that have a polar and an uncharged tail. This structure is something like a 2-dimensional liquid. It is relatively elastic and allows uncharged molecules that are not too big to path quite well. Since DNA is huge and strongly charged it normally won’t pass easily. To pass this barrier you have to torture your yeast a bit (*laughs manically in evil scientist). You submit them to a sudden increase of temperature to 42 °C from room temperature. Doesn’t sound too bad but yeast likes it best between 20 and 30 °C above 40 °C starts killing them slowly. This is thought to built differences in pressure between the hot outside and cool inside of the cell which is equalized by the formation of pores in the membrane. Through these pores the DNA can now enter the cell.
To avoid another line of defense of the cells which is enzymes that eat DNA, you also add some carrier DNA, which will be eaten first and so protects the DNA you want to get into the cells. As carrier DNA you normally use Salmon Sperm that you boil and then add to the mix. So if you hear molecular biologists talk about cooking some sperm it’s probably not as weird as it might sound.
After overcoming the defenses you now have a lot of dead cells, many that have taken up nothing or only the carrier DNA but you hopefully also have some that have the DNA that you wanted to introduce into them. What happens with this DNA depends on its form. If it is a closed circle and includes some special signals in the code it will be just kept in the cell and will be copied to the daughter cells when the cell replicates. This is called a plasmid and is good if you just want to introduce genes more transiently without permanently changing the genome. If the DNA (like in my case) is a linear open molecule produced by PCR and has ends that resemble a sequence in the genome of the yeast, it can go into the nucleus of the cell, where the genome is kept. There it can jump in while the cell replicates and integrate permanently into the genome.
Now you have to filter for those cells where this has worked from the rest. You do this with selection. You seed your cells to a medium where only those cells can grow that have taken up your DNA. This is done by adding a marker to the DNA which is a gene that either makes your cells resistant to something that is poisonous to the others or that let’s them live of a food source that the others can’t use. I used cells that normally can’t make Uracil (which is a part of DNA) themselves so they can’t grow on medium where there is no Uracil. They only can grow on this medium if they have taken up my construct which includes a gene that helps them make their own Uracil.
The problem is that this integration is very rare. You start with a huge amount of cells, transform them seed them all to the selection plate and sometimes get nothing, sometimes one or two colonies but sometimes, if you’re lucky, you also get something like 100.
To verify that the construct is really integrated where I hoped it would, I now need another PCR. I chose primers that normally produce a short fragment of DNA from the region where I want to integrate my construct. If it is successfully integrated the fragment gets longer which I can see in a gel electrophoresis. And that is what I’m currently doing. I’ll soon give you an update when I’ve finally found my aging yeast.
Teaching Yeast to Age like normal People Part III
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In the post “Teaching Yeast to Age Part II” I explained what I tried first to teach my yeast to shorten its Telomers at each cell division. I also told you that it did not work yet. While I have not yet completely given up on Tretrad dissection, I decided to first try another way. It would already be nice to have my fluorescently tagged cells with a knocked out Telomerase but it would be a lot cooler to be able to switch the Telomerase on and off at will.
The way I’m trying to achieve this is called promoter shuffle. As you might know, Proteins (like the subunit of Telomerase I’m interested in) are encoded in the genome by a fixed code where 3 letters (called a codon) in the DNA (bases) are translated into one letter in the protein (amino acid). This code is well known and understood. All codes for proteins start with the same start codon and end with one of three stop codons. However to really produce this protein in the cell there needs to be more. The gene needs to be flanked by regulatory code that influences if and how strongly the encoded protein is produced. Most important for this is the so called promoter region. This is the code directly before the start codon. It is normally between 50 and a few hundred bases long. Their code is a lot less understood than that for the protein. There are strong and weak promoters, promoters that are always active and promoters that are switched off or on on specific stimuli.
Since I want to make the Telomerase switchable I take the genetic code for one of its subunits and exchange some of the code before its start codon against something else. There are a handful of common switchable promoters in yeast that are well described but most of them have some disadvantages. I identified a relatively newly identified one that I hope will be a good choice.
It is the promoter of the gene DDI2 that is normally almost inactive but increases its activity more than 1000-fold if the cells are incubated with the chemical cyanamide. In the concentration it is used here cyanamide has almost no effect on the yeast cells.
Exchanging code in the yeast genome luckily isn’t too complicated. Yeast quite readily incorporates genetic material that you feed it if a stretch at the beginning and end of the inserted code match the code in the genome. This process is called recombination. Recombination is also the process that makes children unique instead of copies of their parents because it randomly mixes the genetic material of your parents that is passed on to your kids.
So I need a stretch of DNA containing the 500 bases before the start codon of DDI2 with 50 bases on both ends from the area before the start codon of the Telomerase subunit. I did a lot of thinking how to build this myself but it would have been a lot of work. Then I thought about the paper where I first read about the DDI2 promoter. In the nature article “Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast” Professor Yu Fu of the Chinese Academy of Sciences and colleagues characterized the promoter and explained how they integrated it in genomic locations.
So I just wrote an email to Professor Fu asking if I could get some of the material they used. He actually replied within a day and said that he’d be happy to help me. He sent me the code of the promoter without expecting anything in return. That’s a great thing about the science community. Most people are happy to share and are really nice if you show interest in their work.
So all I have to do now is add the code stretches at the ends that determine the target where I want to integrate it. I’m doing this with a PCR which I’ll explain in my next article.
What are Telomeres?
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To explain what I’m doing I’ll first explain what Telomeres are and why their length is relatively important.
Telomeres are solving a problem that came up when bacteria became eucariots (plants, mushrooms and animals). At this point they stopped storing their genetic information on DNA-rings and instead developed linear chromosomes. This brings some structural advantages for bigger chromosomes but has a huge disadvantage. Each time a cell divides (which most cells do a lot) it has to replicate it’s genome so each daughter can have a copy. This is done by enzymes called DNA-polymerases. The process only works in one direction on the DNA-string and doesn’t start at the beginning.
For a circular genome this is no problem but for a linear genome this means the loss of a few bases (the building blocks of DNA) at each devision. For a while this is not a huge problem if you don’t write important stuff at the beginning (or end) of the chromosome. And that is already the first function of Telomeres. Aside from mechanically stabilizing the chromosome ends, they are a peace of genetic code at the ends of chromosomes that do not contain vital information.
This is already enough for many of our somatic cells (those cells in the body that form most of our body) they only divide a certain number of times until a tissue (like a muscle or your brain) is formed and then just work and do not or only rarely divide.
Only cells that need to divide unlimited or often (germ line or stem cells but also unicellular life) need another trick. This trick is called Telomerase and earned it’s discoverers a Nobel Prize. Telomerase is an enzyme complex that uses an RNA-template to elongate Telomeres and so completely solve the so called End-Replication-Problem.
The fact that most somatic cells are not using this trick and have telomerase switched off has an interesting implication. It makes Telomeres something like a cellular clock of aging. They limit the number of times a cell can divide and rejuvenate a tissue before it goes into a state called senescence. The more cells of an organ are in this state, the less likely the whole organ gets to repair itself and to function correctly.
Another type of cell that needs to replicate a lot is cancer. This is why about 4 out of 5 cancer cells have Telomerase switched on.
The full picture of the connection between Telomeres, aging and cancer are quite complicated and still under discussion but it is quite clear that influencing Telomere length has quite some potential to treat cancer on one side and influence aging on the other.
If you’re interested in a good overview on the current state of knowledge on the connection between aging and Telomeres there’s a quite new review article from Vaiserman et al., 2021.
If you want further information on Telomers and cancer I’d recommend the article of Hiyama et al., 2003 about Telomerase as tumor marker or Graham et al., 2017 about Telomeres and prostrate cancer.
For an overview on Telomeres as a target for drug discovery see the nature article of Bearss et al., 2000.