Dissection of Saccharomyces Cerevisiae Asci

Summary

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.

Abstract

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.

Protocol

Construction of diploid strains
Protocol for production of diploids by mating

1. 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 MATα).
2. 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 arrow on the bottom of the plate.
3. Repeat this for the second strain on the same YPD plate. This time streak the colony perpendicular to the first, cros....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Singer MSM System 200	Microscope	Singer Instruments	NA
D-sorbitol	Reagent	BioShop Canada	SOR508
Tris	Reagent	BioShop Canada	TRS001
Zymolyase 100T	Reagent	Seikagaku Corporation	120493
Yeast extract	Reagent	BioShop Canada	YEX401
Peptone	Reagent	BioShop Canada	PEP 403
D-glucose	Reagent	BioShop Canada	GLU501
Yeast nitrogen base	Reagent	BioShop Canada	YNB406
Bio-tryptone	Reagent	BioShop Canada	TRP402
Sodium chloride	Reagent	BioShop Canada	SOD002
Potassium acetate	Reagent	BioShop Canada	POA 301
Agar	Reagent	BioShop Canada	AGR003
Adenine sulfate	Reagent	BioShop Canada	ADS201
L-uracil	Reagent	BioShop Canada	URA 241
L-tryptophan	Reagent	BioShop Canada	TRP 100
L-histidine	Reagent	BioShop Canada	HIS 200
L-arginine	Reagent	Amresco	0877-100G
L-methionine	Reagent	BioShop Canada	MET 222
L-tyrosine	Reagent	BioShop Canada	TYR 333
L-isoleucine	Reagent	BioShop Canada	ISO 910
L-valine	Reagent	BioShop Canada	VAL 201
L-lysine	Reagent	BioShop Canada	LYS 101
L-phenylalanine	Reagent	BioShop Canada	PHA 302
L-glutamic acid	Reagent	BioShop Canada	GLU 202
L-threonine	Reagent	BioShop Canada	THR002
L-leucine	Reagent	BioShop Canada	LEU 222