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Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.
Note that the following protocol is described for a Singer MSM System 200 micromanipulation microscope. This protocol can be followed with slight modifications if using a manual micromanipulator such as a Zeiss micromanipulator MR.
Figure 1: The mating of two haploid strains of opposite mating types generates a diploid strain.
Figure 2: (A) A YPD plate with thre results of a mating between a temperature sensitive strain (with a leu2 auxotrophy) and a wild type strain (LEU2). Digested cells are spread in the inoculum region on the left where thick growth between the two black lines is seen. Asci that have been dissected grow evenly spaced in the matrix on the right. (B) The plate in (A) was replicated onto an SD -Leucine drop-out plate. Note the 2:2 growth for the LEU2 marker indicating a validated ascospore was dissected. (C) The plate in (A) was replicated onto a YPD plate and incubated at 37°C. Note the 2:2 growth that further validates that an ascospore was dissected.
Yeast dissection is a useful tool to select new strains with desired markers. When determining the theoretical growth pattern of four ascospores it is important to look at the genotype of the vegetative cell. If a plasmid is present, one must know if it is single, low or high copy as this will affect which ascospore(s) receive the plasmid and will affect predictions and conclusions regarding the strains being manipulated.
Certain strains can sporulate better than others producing many ascospores in a short period of time. Other strains may only yield a low percentage of ascospores. This does not have any affect on the experiment as long as enough true asci can be selected. Furthermore, strains respond differently during cell wall digestion and it is necessary to empirically adjust the incubation time in the tetrad juice.
When selecting cells with the desired markers, one should only choose those that come from an asci where the growth pattern of all ascospores correspond to the theoretical growth pattern and from a dissection where the vast majority of all ascospores lead to the expected growth pattern.
Work in the author’s laboratory is funded by the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council and Concordia University.