Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.     

PET genes of Saccharomyces cerevisiae.

We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae.     

Mating-type gene switching in Saccharomyces cerevisiae

The study of yeast mating-type (MAT) gene switching has provided insights into several aspects of the regulation of gene expression. MAT switching is accomplished by a highly programmed site-specific homologous recombination event in which mating-type-specific sequences at MAT are replaced by alternative DNA sequences copied from one of two unexpressed donors. The mating-type system has also provided an opportunity to study both the genetic regulation of gene silencing by alterations in chromatin structure, and the basis of preferential recombination between a recipient of genetic information and one of several possible donors.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae

We have detected two site-specific endonucleases in strains of Saccharomyces cerevisiae. One endonuclease, which we call YZ endo, is present only in yeast strains that are undergoing mating-type interconversion. The site at which YZ endo cleaves corresponds to the in vivo double-strand break occurring at the mating-type locus in yeast undergoing mating-type interconversion. YZ endo generates a site-specific double-strand break having 4-base 3' extensions terminating in 3' hydroxyl groups. The site of cleavage occurs in the Z1 region near the YZ junction of the mating-type locus. Mutant mating-type loci known to decrease the frequency of mating-type interconversion are correspondingly poor substrates for YZ endo in vitro. In vitro analysis of a number of such altered recognition sites has delimited the sequences required for cleavage. The molecular genetics of mating-type interconversion is discussed in the context of this endonucleolytic activity. The second endonuclease, which we refer to as Sce II, is present in all strains of S. cerevisiae we have examined. The cleavage site of Sce II has been determined and proves to be unrelated to the cleavage site of YZ endo.     

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.     

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.     

Intermediates of recombination during mating type switching in Saccharomyces cerevisiae.

We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.     

Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging

The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast.     

Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.