Tag: moleculat biology

Teaching Yeast to Age Part I

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In my last article I explained the aim of my project, which is making Telomere length of yeast visible. I also explained the first problem for this: Yeast holds its Telomers constant by a tightly controlled use of Telomerase. That means it doesn’t show Telomere attrition as a sign of aging like our somatic cells do.

This is why I have to teach yeast to age like normal people first. The most obvious strategy to do this is to switch off their Telomerase. In molecular biology you normally do this by removing or destroying the gene or in this case one of the genes that code for Telomerase. We call this a knock-out mutation or just knock-out.

One of the big advantages of yeast as a model organism is, that there are libraries of yeast strains available where you can not only find yeast strains with any protein tagged with Green fluorescent protein (as I explained last time) but also strains with any gene knocked-out. An exception are obviously genes that are so vital that knocking them out makes the yeast in-viable. The genes associated with Telomerase are a bit special in this because yeast with one of them knocked-out is viable but only for a certain number of generations (about 400). One generation under ideal circumstances is about 90 minutes for yeast. So after cultivating them for about 4 weeks their first Telomere become critically short and their cell cycle is stopped permanently. They go into a phase of senescence like our somatic cells at that point.

So basically it would be easy to just thaw one of these knock-out strains from our – 80 °C freezers and I’ve got yeast that ages. The problem however is that I also need a mutation from another library. As I explained in the last article I want to use Telomere associated proteins that are fused with Green fluorescent protein as a marker. So I’ve got a few options: I can take a strain with one mutation and do the other mutation myself. I’ll tell you another time how that works.

Or I can bring both mutations into one cell by mating the two strains. Now I have to explain another cool feature of yeast as a model organism. You might have heard that all the genetic information in your cells is kept in two versions. One from your father and one from your mother. This is called diploid. It has some advantages to have this. The most obvious is that if an important gene is broken on one of your chromosomes there is still a second copy of it from your other parent that can fix it. On the other side in our germline cells (eggs and sperm cells) there is only a single copy of each chromosome. This is made by randomly mixing the the chromosomes you’ve got from your parents. This is also why each egg and each sperm is genetically different. This is called haploid. This form is obviously practical for sexual reproduction because it stops us from collecting more and more copies of the same information in our cells. But it is also great for me as a molecular biologist because if I want to switch off a gene I only have to do so once. There is no second copy that could make trouble.

Now comes the great thing about yeast: It can stably live in a haploid and a diploid form. There is no male or female yeast but there is something similar: The mating types Mat a and Mat alpha. Haploid (only one set of chromosomes) yeast can be either Mat a or Mat alpha and can only mate with the other mating type upon which they produce diploid offspring and stay diploid. Diploid yeast is both Mat a and alpha but not at the same time. It normally changes it’s sex every time it produces offspring. Diploid yeast only mates with diploid and not with haploids even it it currently has the opposite sex. To make diploid yeast haploid again they need to sporulate. Spores are a dormant form of yeast that the produce in bad times. In the lab you create bad times for them by first giving them ample sugar and than suddenly putting them on a very strict diet.

So how does this all help me produce a haploid yeast strain with mutations from two different strains? I first cross the two mutant strains of opposing mating type from which I get a diploid strain with one of the mutations on one set of its chromosomes, the other mutation on the other. Then I bring them to sporulate by setting them on a diet. In the process of sporulation one diploid cell produces four genetically different haploid spores by randomly sharing parts of their chromosomes from each of their two parents. These four spores stay together in one bag (called the Ascus) until times get better and they grow out again and mate. Now if I’m lucky one of these 4 spores has both of the mutations I want in it.

Now the big trick is finding it and this is done by sorting single tiny yeast cells. I’ll show you this crazy technique next time.

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