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Tomitaka M, Taguchi H, Matsuoka M, Morimura S, Kida K, Akamatsu T J Biosci Bioeng. 2014 Jan;117(1):65–70 We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploidstrains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies. The first strategy was as follows: (i) the KF7-5C was crossed with the laboratory strain SH6710; (ii) the progenies were backcrossed with KF7-5C three times; and (iii) the progenies were inbred three times to maintain a genetic background close to that of KF7. The NAM12 diploid between the cross of the resultant two strains, NAM11-9C and NAM11-13A, showed efficient sporulation and exhibited excellent growth in YPD medium (pH 3.5) at 35°C with 1.4-h generation time, indicating thermotolerance and acid tolerance. The second strategy was successive intrastrain crosses. The resultant two strains, KFG4-6B and KFG4-4B, showed excellent mating capacity. A spontaneous mutant of KFG4-6B, KFG4-6BD, showed a high growth rate with a generation time of 1.1 h in YPD medium (pH 3.0) at 35°C. The KFG4-6BD strain produced ascospores, which were crossed with NAM11-2C and its progeny to produce tetrads. These tetrads were crossed with KFG4-4B to produce NAM26-14A and NAM26-15A. The latter strain had a generation time of 1.6 h at 35°C in pH 2.5, thus exhibiting further thermotolerance and acidtolerance. A progeny from a cross of NAM26-14A and NAM26-15A yielded the strain NAM34-4C, which showed potent lactic acid assimilation and high transformation efficiency, better than those of a standard laboratory strain.

Louvel H, Gillet-Markowska A, Liti G, Fischer G Yeast. 2014 Mar;31(3):91–101 Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S. cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species. The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European and Sake) and represent clean, non-mosaic lineages of S. cerevisiae, meaning that their genomes differ essentially by monomorphic and private SNPs. In this study, one representative strain for each of the five S. cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions, so that all dominant markers remained available for further genetic manipulations. A set of 50 strains was assembled, including eight haploid and two diploid strains for each lineage. These strains carry different combinations of leu2∆0, lys2∆0, met15∆0, ura3∆0 and/or ura3∆::KanMX-barcoded deletions with marker configurations resembling that of the BY series, which will allow large-scale crossing with existing deletion collections. This new set of geneticallytractable strains provides a powerful tool kit to explore the impact of natural variation on complex biological processes.

Fernández-González M, Úbeda JF, Briones AI Curr Microbiol. Springer US; 2015 Mar;70(3):441–9 One of the issues that most concerns to both winemakers and producers of active dry yeasts is the stuck and sluggish fermentations of grape musts with high levels of sugar, reflecting the inability of inoculated yeast strain to complete the fermentation process. It is difficult to obtain a winestrain that possesses both adequate oenological and technological properties; thus, the correct approach to solving these problems is the application of breeding programs primarily focused on both properties. The first step toward this process is to characterize the phenotypic diversity between potential parental strains. In the present study, we have analyzed the fermentative behavior of 26 Saccharomyces cerevisiae wine strains in high-sugar conditions at 20 °C, using a range of tests, such as sporulation ability, spore viability, and tetrad analysis to determine the tolerance of these yeasts to several stress conditions. Most tested strains were homothallic and heterozygous for more than one character. Two auxotrophic derivatives with defects in amino acid or nucleic acid metabolism were obtained, and these strains could potentially be used for the development of hybridization techniques without using laboratory strains.

Yan Z, Xu J Genetics. 2003 Apr;163(4):1315–25 Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from sixcrosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.

Reverter-Branchat G, Cabiscol E, Tamarit J, Sorolla MA, Angeles de la Torre M, Ros J Microbiology (Reading, Engl). 2007 Nov;153(Pt 11):3667–76 Alcohol dehydrogenase 1 (Adh1)p catalyses the conversion of acetaldehyde to ethanol, regenerating NAD+. In Saccharomyces cerevisiae, Adh1p is oxidatively modified during ageing and, consequently, its activity becomes reduced. To analyse whether maintaining this activity is advantageous for the cell, a yeast strain with an extra copy of the ADH1 gene (2xADH1) was constructed, and the effects on chronological and replicative ageing were analysed. The strain showed increased survival in stationary phase (chronological ageing) due to induction of antioxidant enzymes such as catalase and superoxide dismutases. In addition, 2xADH1 cells displayed an increased activity of silent information regulator 2 (Sir2)p, an NAD+-dependent histone deacetylase, due to a higher NAD+/NADH ratio. As a consequence, a 30% extension in replicative life span was observed. Taken together, these results suggest that the maintenance of enzymes that participate in NAD+/NADH balancing is important to chronological and replicative life-span parameters.

Smith GR Methods Mol Biol. 2009;557:65–76 The fission yeast Schizosaccharomyces pombe is well-suited for studying meiotic recombination. Methods are described here for culturing S. pombe and for genetic assays ofintragenic recombination (gene conversion), intergenic recombination (crossing-over), and spore viability. Both random spore and tetrad analyses are described.

Furuya K, Niki H Yeast. 2009 Apr;26(4):221–33 The fission yeast Schizosaccharomyces japonicus var. japonicus belong to the genus Schizosaccharomyces, together with Schizosaccharomycespombe, which has been well studied as a model organism. In contrast, Sz. japonicus is poorly characterized and genetic tools were yet to be developed. We here report the isolation of the heterothallic haploids NIG2017, NIG2025 and NIG2028, which were derivatives of a Sz. japonicushomothallic strain (NIG2008). Based on the genomic sequence of Sz. japonicus, released by the Broad Institute, we found that Sz. japonicus also possesses orthologues of the mating-type genes of Sz. pombe; two mat-M (-) and two mat-P (+) genes. As expected, heterothallic strains were defective in one of the Sz. japonicus mat genes (mat(sj)). We confirmed that NIG2017 and NIG2025 strains only expressed mRNA from the mat(sj)-P genes, while homothallic strains expressed both mat(sj)-M and mat(sj)-P. Although the NIG2028 strain expressed both gene products, mat(sj)-P was found mutated, which may have conferred the heterothallic phenotype of the mutant. Thus, we concluded that these were stable heterothallicstrains. We designated NIG2017 and NIG2025 as h(+) and NIG 2028 as h(-), respectively. We also found additional h(-) strains (NIG5872 and NIG5873) that arose from the cross between NIG2017 and NIG2028 derivatives. In addition to that, we have constructed a ura4(sj)-deleted strain and an ade6(sj)-mutated strain. We used these heterothallic strains and the auxotroph strains to perform spore dissection analysis to determine the genetic distances between several loci, and found that the mating type loci and ade6(sj) locus were linked to centromeres.

Moreno JI, Buie KS, Price RE, Piva MA Curr Genet. 2009 Aug;55(4):475–84 YGR150C gene product (Ygr150cp) is one of the three mitochondrially located Saccharomyces cerevisiae proteins with pentatricopeptide repeat(PPR) motifs. Ygr150cp is essential for mitochondrial functionality but its molecular targets are still unknown. This study was undertaken to define the role of Ygr150cp in mitochondria biogenesis. Repression of Ygr150cp expression in complemented mutants prevented their use of glycerol or lactate, but allowed limited growth on ethanol-containing medium. RNA hybridization studies showed that Deltaygr150c meiotic segregants producedCOB and COX1 transcripts but failed to process them into the mature forms. Detailed RT-PCR assays revealed that Deltaygr150c specifically failed to remove the fourth intron of both COB and COX1 pre-mRNAs while all other group I introns were excised. Expression of Ygr150cp mutants without any of the PPR motifs did not complement the growth phenotype. Accordingly, we designate YGR150C as CCM1 (COB and COX1 mRNA maturation). This report provides the first evidence of PPR protein involvement in the specific removal of group I introns in mitochondria of S.cerevisiae.

Carter Z, Delneri D Yeast. 2010 Sep;27(9):765–75 In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre-loxP system, so that they can be used withnatural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE-hphNT1-loxRE and lox2272-natNT2-lox2272 gene-disruption cassettes can be present in the yeast genome together with the widely used loxP-marker gene-loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin-resistant Cre-expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon-limited resources.

Yewdell WT, Colombi P, Makhnevych T, Lusk CP Mol Biol Cell. 2011 Apr 15;22(8):1375–88 Nuclear pore complexes (NPCs) provide a gateway for the selective transport of macromolecules across the nuclear envelope (NE). Although we have a solid understanding of NPC composition and structure, we do not have a clear grasp of the mechanism of NPC assembly. Here, we demonstrate specific defects in nucleoporin distribution in strains lacking Heh1p and Heh2p-two conserved members of the LEM (Lap2, emerin, MAN1) family of integral inner nuclear membrane proteins. These effects on nucleoporin localization are likely of functional importance as we have defined specific genetic interaction networks between HEH1 and HEH2, and genes encoding nucleoporins in the membrane, inner, and outer ring complexes of the NPC. Interestingly, expression of a domain of Heh1p that resides in the NE lumen is sufficient to suppress both the nucleoporin mislocalization and growth defects in heh1Δpom34Δ strains. We further demonstrate a specific physical interaction between the Heh1p lumenaldomain and the massive cadherin-like lumenal domain of the membrane nucleoporin Pom152p. These findings support a role for Heh1p in theassembly or stability of the NPC, potentially through the formation of a lumenal bridge with Pom152p.