Selection ofYeastAuxotrophs byThymidylate Starvation

Nutritional auxotrophs ofSaccharomyces cerevisiae canbereadily obtained byusing chemicalorphysical treatments thatincrease mutationfrequencies toconveniently detectable levels. Onedisadvantage ofthisapproach isthat themutagenic treatments caninduce mutations atseveral loci within asingle cell. Consequently, theyareoflimited useintheconstruction of strains differing ingenotype ata single locus butremaining otherwise isogenic. A procedure forselecting spontaneous auxotrophs byusing inositol-less deathhasbeenemployed withsuccessinsomelaboratories butrequires thatthe inositol markers befirst crossed into anappropriate background (1). Thymnineless enrichment ofauxotrophic mutantsdepends uponthesamegeneral strategy asthat usedintheinositol-less procedure. Growingyeastcells inwhichdeoxythymidine 5'monophosphate (dTMP)biosynthesis isabolished either bymutation (tmpmutants) orby folate antagonists (aminopterin andsulfanilamide) undergo thymineless death(J.G.Little andR.H.Haynes, manuscript inpreparation). Cells harboring mutations thatresult inablock inribonucleic acidorprotein synthesis arerelatively immune.Oneormorecycles ofthymineless killing cantherefore beusedasanintense enrichment forsuchmutants. Since S.cerevisiae strains appear tobegenerally sensitive tothe combination aminopterin plus sulfanilamide, we suppose thatthismethodmay beofgeneral utility. Theselection principle isillustrated inFig. 1, whichshowsthethymineless deathkinetics of a methionine-requiring dTMP auxotroph deprived ofdTMP inminimal mediuminthe presence orabsence ofexogenous methionine. After24h inthepresence ofmethionine, approximately 0.5%ofthecell population remained viable. Intheabsence ofmethionine nocell deathoccurred.