Ribosomal DNA magnification in Saccharomyces cerevisiae.

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.     

The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M₁-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.     

Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae

With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose. Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III. RAP-I was previously characterized. RAP-III is present in a very small amount, which hindered its purification. RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60. RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular mass of around 62 kDa and it is immunologically different from RAP-I and PK60. RAP-II phosphorylates the P proteins in the last serine residue at the highly conserved carboxyl terminal domain as other P-protein kinases. The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases. Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha whereas RAP-I and CK-II modify all of them. A comparative study of the K(m) and V(max) of the phosphorylation reaction by the different kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome. All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements.     

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.     

Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen.

A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. bayanus should be reinstated as a separate taxon.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Initiation of DNA replication in Saccharomyces cerevisiae G1‐phase nuclei by Xenopus egg extract

Xenopus egg extracts initiate replication at specific origin sites within mammalian G1‐phase nuclei. Similarly, S‐phase extracts from Saccharomyces cerevisiae initiate DNA replication within yeast nuclei at specific yeast origin sequences. Here we show that Xenopus egg extracts can initiate DNA replication within G1‐phase yeast nuclei but do not recognize yeast origin sequences. When G1‐phase yeast nuclei were introduced into Xenopus egg extract, semiconservative, aphidicolin‐sensitive DNA synthesis was induced after a brief lag period and was restricted to a single round of replication. The specificity of initiation within the yeast 2 μm plasmid as well as in the vicinity of the chromosomal origin ARS1 was evaluated by neutral two‐dimensional gel electrophoresis of replication intermediates. At both locations, replication was found to initiate outside of the ARS element. Manipulation of both cis‐ and trans‐acting elements in the yeast genome before introduction of nuclei into Xenopus egg extract may provide a system with which to elucidate the requirements for vertebrate origin recognition. J. Cell. Biochem. 80:73–84, 2000. © 2000 Wiley‐Liss, Inc.     

Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae

NAD⁺ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD⁺ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD⁺ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD⁺-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD⁺ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD⁺ precursors. Together, our studies provide a molecular basis for how NAD⁺ homeostasis factors confer metabolic flexibility.The pyridine nucleotide NAD⁺ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD⁺ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (1, 2). NAD⁺ also plays an important role in calorie restriction (CR)²-mediated life span extension via regulating NAD⁺-dependent longevity factors (3, 4). CR is the most effective regimen known to extend life span in various species (5, 6). CR also ameliorates many age-related diseases such as cancer and diabetes (5). The Sir2 family proteins are NAD⁺-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (3, 7) and higher eukaryotes (8, 9). By coupling the cleavage of NAD⁺ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (8–10), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging.In eukaryotes, NAD⁺ is generated by de novo synthesis and by salvaging various intermediary precursors (see Fig. 1A). In yeast, the de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (11). Because the de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD⁺ precursors for the nicotinamide (Nam) moiety (11). Yeast cells also salvage Nam from NAD⁺ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD⁺ via Nma1/2 and Qns1 (see Fig. 1A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (12, 13), and Qns1 is a glutamine-dependent NAD⁺ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD⁺ auxotrophic mutants (14–16). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (14, 16). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (15, 16).Open in a separate windowFIGURE 1.Nicotinamide riboside (NmR) is an endogenous metabolite in yeast. A, the current model of the NAD⁺ biosynthesis pathways. Extracellular NmR enters the salvage cycle through Nrk1, Urh1, Pnp1, and Meu1. B, NAD⁺ prototrophic cells release metabolites into growth medium to cross-feed NAD⁺ auxotrophic cells (the npt1Δqpt1Δ and qns1Δ mutants). Micro-colonies of the NAD⁺ auxotrophic mutants become visible after 2-day incubation at 30 °C, which show “gradient” growth patterns descending from the side adjacent to WT. C, Nrk1 is required for NAD⁺ auxotrophic cells to utilize NmR. Anaerobic growth conditions (−O₂) are utilized to block de novo NAD⁺ biosynthesis in the npt1Δ and npt1Δnrk1Δ mutants. D, Nrk1 is required to utilize cross-feeding metabolites. E, cross-feeding activity is modulated by factors in NmR metabolism. Cells defective in NmR utilization (left panel) or transport (middle panel) show increased cross-feeding in spot assays. Overexpressing Nrk1 decreases cross-feeding activity (right panel). The results show growth of the npt1Δqpt1Δ recipient (plated on YPD at a density of ∼9000 cells/cm²) supported by feeder cells (∼2 × 10⁴ cells spotted directly onto the recipient lawn). oe, overexpression.NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (17). For example, NmR protected neurons from axonal degeneration via functioning as a NAD⁺ precursor (18, 19). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (14, 15, 20–22), NmR has been speculated to be an endogenous NAD⁺ precursor (17, 23). Here, we provided direct evidence for endogenous NmR as an integral part of NAD⁺ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD⁺ levels in mammalian cells (24). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes.     

腺甘酸环化酶Cyr1突变对酿酒酵母时序寿命的影响

为了研究腺甘酸环化酶Cyr1对酵母细胞时序寿命的影响,我们使用Cyr1△与野生菌DBY746来检测酵母细胞的时序寿命,H_2O_2或热抵抗能力以及线粒体膜形态。结果表明,Cyr1△的时序寿命是DBY746的2.25倍,在强卡路里限制调节下,两者的生存时间均有延长,DBY746和Cyr1△达到10%生存率的时间分别为13d和45d。Cyr1△有更强的抵抗H_2O_2和热处理能力,同时发现Cyr1△比DBY746在培养第3d和第5d有更完整的线粒体膜形态。     

Cell Wall Assembly in Saccharomyces cerevisiae

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.