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Dissection Microscope
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Micromanipulator
LMO Bordeaux 2014
 

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Yeast Genetics

Summary

Yeasts are single-celled, eukaryotic organisms (eukaryotes are organisms with a nucleus containing their genetic material). Because yeasts are evolutionarily related to humans in many ways, they are often used as a model system for how things work in our own cells. The similarity between many yeast and human proteins, along with the ease of handling and experimenting with yeast makes it a popular model to study. Two very common types yeast used in research are Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Storage and Handling

Yeast strains are often stored in small plastic tubes or on plates in the freezer. Small amounts can be picked and expanded in liquid media or streaked onto agar plates using a sterile wire loop. Yeast are typically grown at a lower temperature than mammalian cells, as their ‘cellular machinery’ has evolved to operate and function between 28-32oC. You can see a video on how to grow yeast here.

The advantages of Yeast

Yeast is easy to handle, safe to use, cheap and simple to grow, and many experiments can be performed quite rapidly. In addition, yeast is extremely genetically tractable, and well-established tools are available for manipulating it’s genetics. Finally, yeast has perhaps the largest collection of resources available relating to the function of its genes as well as complete libraries of knockout strains.

Yeast life-cycle

Under normal conditions yeast undergoes vegetative growth. This is the non-sexual stage of the life-cycle, involving growth, metabolism, DNA replication and cell division. In the lab, yeast can be one of two mating types – type a or type α (alpha) - analogous to male and female types. When these types are grown together they release chemicals that attract one another, then fuse together and exchange genetic material; this is the sexual part of the yeast life-cycle. Both parts of the yeast life-cycle are exploited in genetic research to study a variety of biological processes.

Storing Yeast

Yeast Genetics

We can mate different strains of yeast. Mating (or crossing) yeast allows researchers to produce strains with new genetic combinations or mutations. This helps understand the effects of genetic interactions, and how different proteins work together. When yeast strains are crossed a phenomenon called recombination can occur. This is a natural process in which genetic material (on chromosomes) from one strain gets ‘swapped’ with the other strain. This may or may not happen spontaneously, but from the researcher’s perspective it is desirable to know if it has happened, as it has a bearing on which cells to select for their on-going experiments.

The Singer RoToR robot can rapidly pin thousands of different yeast strains on top of one another to allow the yeast to mate. The RoToR can be seen in action here. Mated colonies can then be transferred into new selection conditions that allow only the mated yeast to survive and sporulate.

Sporulation is a process that occurs when cells of mated yeast strains are grown in low-nutrient conditions. The cells divide and form a small sack (or ascus) containing four yeast spores - this is known as a tetrad. Each set of spores may possess a different genetic combination of parental chromosomes, a unique mixture of the original parent chromosomes, or a recombined collection of chromosomes.

Yeast Tetrad Dissection

Following mating and sporulation, researchers can determine which spores carries which genetic elements, but to do this they must separate (dissect) the four spores and grow them as individual colonies. Yeast dissection microscopes such as the Singer MSM 400 have made this process fast and easy – for a video showing how to dissect a tetrad click here.

Essentially, tetrads are plated onto agar dishes which are inverted on the microscope stage to keep the agar plate as sterile as possible whist it is being worked on. A very fine needle tip is elevated from below, which can be seen as it approaches the damp surface of the agar where the yeast tetrad lies. Contact between the needle and the yeast is easily observable as the needle tip touches the wet surface. Retracting the needle tip away from the surface usually ‘picks’ any yeast cell from that spot, the plate can be moved and the yeast deposited in a new position – again by gently making contact with the wet surface.

Spore Analysis

Sometimes gentle tapping of the bench or microscope stage is usually sufficient to separate the spores. A second, more elegant solution is provided on the MSM 400 Series using a Micro Zapper, which produces a vibration that readily separates the spores without damaging them. Automatic grid positioning and a positional recall feature then allows precise arrangement of each spore elsewhere on the plate.

Now the researcher can grow these spores into colonies and decide which carry the correct combination of genes or mutations to choose for their experiements. The selected colony will be expanded in liquid media or streaked on solid agar, then stored for future use or used for experimentation.

Yeast strains can be repeatedly crossed to produce new strains containing several mutations or lacking several genes. The Greek symbol ∆ (delta) denotes a missing or mutant gene, e.g. ∆Scp1∆Mal3∆Eg5.

Yeast Mutants & Screening

The entire yeast genome library is now available to researchers, meaning that any yeast gene can be tested under different conditions. Yeast strains lacking a specific gene will be named by placing the Greek letter delta before the abbreviation for the missing gene; for example Scp1 indicates that this yeast is missing the gene to produce Scp1 protein. The Singer RoToR robot performs the duplication of a large arrays of yeast colonies in a consistent way and with reproducible quality. This saves a huge amount of time compared to doing it by hand. It also allows the printing of a whole library of yeast onto multiple plates, each with a different test condition e.g. different antibiotic, to see which genes help the cells survive. The inclusion of antibiotics, or the deliberate withdrawal of specific nutrients to force only the growth of genetically desirable cells is referred to as selection.

Glossary

Agar / YPD Agar
A solid growth medium containing nutrients, on which yeast and bacteria are grown. Liquid YPD agar is poured into dishes, cools and sets to form a semi-solid surface.
Ascus
The protective membrane that encloses the four spores of a yeast tetrad.
Background
The natural or engineered underlying genetic makeup of an organism.
Crossing
The mating of two strains of organism with different genetic backgrounds.
Dissection
The process of seperating and manipulating the spores of a tetrad.
Libraries
Collections of yeast and bacterial strains each with an individual missing gene or specific genetic mutation.
Mating
The sexual combination of two strains of organism.
Model System
Any organism that is used to study biological processes, which can be compared to those occurring in humans.
Recombination
A natural or engineered process in which organisms rearrange or swap genetic material and integrate it into their own genome.
Reporter Gene
An artificially engineered gene inserted into an organism to allow for selection or visualisation of strains of interest.
Screens
Large-format experiments in which multiple strains are simultaneously tested for sensitivity to a chosen condition, e.g. the presence of an antibiotic.
Selection
The addition or deliberate removal of a chemical to growing cultures, resulting in conditions which will allow only desirable strains to survive and grow.
SGA (Synthetic Genetic Array)
A large-format experiment, in which a selected "query" strain of yeast is crossed with an entire genetic library, in order to study the genetic interactions and the effect on cell survival and growth.
Sporulation
Part of the sexual life-cycle of yeast under low-nutrient conditions, in which mated yeast strains divide and form tetrads.
Tetrad
A collection of four yeast spores enclosed within an ascus, formed following sporulation.
YPD
Yeast extract, Peptone, Dextrose. The basic ingredients of yeast growth media, containing all essential substances to promote microbial growth.