Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format.
During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.
Department of Biology, University of Massachusetts Boston.
Scott M
SM
Paulissen
Department of Biology, University of Massachusetts Boston; Linda.Huang@umb.edu.
Linda S
LS
Huang
D008828:Microbiological Techniques / Q000295:instrumentation* / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000502:physiology*; D013172:Spores, Fungal / Q000254:growth & development / Q000502:physiology
Genetics. 2012 Sep;192(1):73-105
22964838
Eukaryot Cell. 2012 Oct;11(10):1191-200
22611022
Genetics. 2016 Jul;203(3):1203-16
27182947
Genetics. 2014 Jan;196(1):31-63
24395824
Cold Spring Harb Protoc. 2015 Nov 02;2015(11):1003-8
26527763
J Bacteriol. 1977 Oct;132(1):180-5
21160
Cell. 1991 Sep 20;66(6):1239-56
1913808
Nature. 2009 Mar 19;458(7236):337-41
19212322
Mol Biol Cell. 2004 Apr;15(4):1802-15
14742704
Nat Methods. 2008 Mar;5(3):239-41
18246075
Annu Rev Genet. 2014;48:187-214
25421598
Curr Opin Microbiol. 2011 Dec;14(6):676-81
21962291
Cold Spring Harb Protoc. 2015 Oct 01;2015(10):908-13
26430251
Appl Environ Microbiol. 1995 Feb;61(2):630-8
7574601
Microbiol Mol Biol Rev. 2012 Mar;76(1):1-15
22390969
Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12431-6
16116083
Methods Mol Biol. 2009;557:21-6
19799173
Genetics. 2011 Nov;189(3):737-65
22084423
J Vis Exp. 2011 Jul 27;(53):null
21841759