Dissection of <em>Saccharomyces Cerevisiae</em> Asci

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.

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot–Marie–Tooth disease model

“…p426-HARS-YFP plasmids were transformed into the diploid ∆HTS1 heterozygous deletion strain and sporulated as described ( 48 ). The spore colonies were genotyped by plating on SD plates lacking uracil to check for the expression of the 2-micron p426 plasmids and on YPD-G418 (350 μg/ml) to select for haploids containing the KanMX gene.…”

SIR telomere silencing depends on nuclear envelope lipids and modulates sensitivity to a lysolipid drug

“…Strains expressing DsRed-HDEL were transformed with the integrating plasmid pAM41 using the standard transformation protocol (Guthrie and Fink, 1991). Strains VZY452, JC4509 and JC4881 were created by mating and tetrad dissection, as described previously (Morin et al, 2009). Briefly, parental strains of opposite mating types were streaked on YPD with one intersecting area to allow mating and produce diploids.…”

Sexually-Driven Combinatorial Diversity in Native Saccharomyces Wine Yeasts

“…To test sporulation capability strains were incubated at 30 • C for 5 days in SPA media and inspected with a microscope. Tetrad dissections were performed according to [71].…”