Directionality of yeast mating-type interconversion

The mating-type a and α alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HMLα HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMRα arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches—that is, MATa to MATa and MATα to MATα—and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that a cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HMLα HMRa strains generate predominantly heterologous while HMLa HMRα strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the Inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.     

Precise Mapping of the Homothallism Genes HML and HMR in SACCHAROMYCES CEREVISIAE

The HML and HMR loci carry unexpressed copies of MATa and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).     

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Illegal transposition of mating-type genes in yeast

Mating-type switching in the yeast Saccharomyces cerevisiae involves the transposition of a copy of a or α information from unexpressed “library” genes, HML or HMR, to replace the sequence at the mating type locus, MAT. In normal homothallic strains, where conversions of MAT may occur as often as every cell division, the switching of MAT alleles does not alter the alleles at HML or HMR. We have discovered that several mutations within or adjacent to MAT that impair the excision of the MAT allele permit conversions of the alleles at HML or HMR in more than 1% of the cells analyzed. The two mutations within the MAT locus (MATa-inc and MATα-inc) can transpose to HML or HMR without being lost at MAT. Thus a MATα-inc HMLα HMRa HO strain can switch to MATα-inc HMLα HMRα-inc HO. Even though the α-inc and a-inc alleles prevent their own replacement at MAT, these sequences are efficiently transposed back from HMLα-inc or HMLa-inc to replace normal MAT alleles. When these alleles reappear at MAT, they are again blocked in excision. Thus the sequences used to remove an allele from MAT must differ from those used to replicate and transpose it. Two cis-acting stk mutations adjacent to MAT that block switching of MATa to MATα also induce the conversion of HMLα to HMLa. However, we have previously shown that these events do not occur in strains carrying a recessive “switch” mutant (swi1) or in strains carrying a defective allele of the HO gene. In stk1 MATa HO strains, HMLα was converted to HMLa in approximately 4% of the subclones examined. In contrast, the HMLα-inc sequence was not converted in similar stk1 MATa HO strains. Thus the excision of the α-inc sequence seems to be prevented at both MAT and HML. These results suggest that the illegal conversions of HML and HMR occur by a mechanism similar to that used for normal conversions of MAT.     

Interconversion of Yeast Cell Types by Transposable Genes

The a and α cell types of budding yeast Saccharomyces cerevisiae are controlled by alternate alleles of the mating-type locus (MAT), MATa and MATα. The cell types can be interconverted by switching alleles of MAT. The loci HMRa and HMLα, which are loosely linked to MAT, are involved in mating-type switching. Experimental evidence for their role in MAT interconversion is presented. As a result of switching, the homothallic and heterothallic strains containing the amber and ochre mutations within the HMRa locus yield corresponding amber and ochre mutant mata loci. Similarly, the hmlα mutant strain generates matα mutant alleles. That is, specific mutations from HMRa and HMLα are transmitted to MAT. A replica of the mating-type coding information originating from these loci is transposed to MAT, where it replaces the existing information. Furthermore, "Hawthorne deletions" in strains containing hmra-amber/ochre result in production of mata-amber/ochre alleles. Therefore, genetic information for MATa resides at HMRa. The switches occur in a defined set of clonally related cells. Thus, the efficient interconversion of yeast cell types is mediated by an unidirectional transfer of genetic information between nonallelic sites in a nonrandom and programmed fashion. The results are inconsistent with the "flip-flop" models, but satisfy a key prediction of the general controlling element and the specific cassette models proposed for mating-type interchange.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

Molecular characterization of β-fructofuranosidases from Rhizopus delemar and Amylomyces rouxii

Of the 19 strains of Rhizopus delemar deposited as Rhizopus oryzae, seven of them, NBRC 4726, NBRC 4734, NBRC 4746, NBRC 4754, NBRC 4773, NBRC 4775, and NBRC 4801, completely hydrolyzed exogenous sucrose and fructooligosaccharides. The sucrose-hydrolyzing enzyme was purified from the culture filtrate of R. delemar NBRC 4754 and classified to β-fructofuranosidase, similar to that of Amylomyces rouxii CBS 438.76. Fragments including β-fructofuranosidase genes (sucA) of seven strains of R. delemar and A. rouxii CBS 438.76 were amplified and sequenced by PCR with degenerated primers synthesized on the basis of the internal amino acid sequences of purified enzymes and successive inverse PCR. Nucleotide sequences of the obtained fragments revealed that open reading frames of 1,569 bp have no intron and encode 522 amino acids. The presumed proteins contained the typical domain of the glycoside hydrolase 32 family, including β-fructofuranosidase, inulinase, levanase, and fructosyltransferases. Amino acid sequences of SucA proteins from the seven strains of R. delemar were identical and showed 90.0 % identity with those of A. rouxii CBS 438.76. A dendrogram constructed from these amino acid sequences showed that SucA proteins are more closely related to yeast β-fructofuranosidases than to other fungal enzymes.     

Design of a minimal silencer for the silent mating-type locus HML of Saccharomyces cerevisiae

The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ΔI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.     

A silencer promotes the assembly of silenced chromatin independently of recruitment

In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.     

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.     

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732T

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732^T) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1''s partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.     

Role of Elg1 protein in double strand break repair

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G₂/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR.