Name: dissection of saccharomyces cerevisiae asci
Uploaded: 2009-05-19T00:00:00
Description: Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

Work in the author&rsquo;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.

Construction of diploid strains 
Protocol for production of diploids by mating
<ol>
	<li>The initial strains should be struck out on YPD, or selective medium if necessary, to produce single colonies. The two haploid strains should be of opposite mating type (ie. MATa and MAT&#945;).</li>
	<li>Using a sterile inoculation loop pick a small portion of a colony from one of the two haploid strains. On a new YPD plate streak the loop once in a straight line across the plate. Mark the direction of the streak with an arro...

<ol>
	<li>Sherman, F., Hicks, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+and+Dissection+of+Asci.">Micromanipulation and Dissection of Asci.</a> Guide to Yeast Genetics and Molecular Biology. , 21-37 (1991).</li></ol>

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 ascospor...

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<td>Singer MSM System 200</td>
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dissection of saccharomyces cerevisiae asci

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.

Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

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