Extensive regulation of metabolism and growth during the cell division cycle

Yeast cells grown in culture can spontaneously synchronize their respiration, metabolism, gene expression and cell division. Such metabolic oscillations in synchronized cultures reflect single-cell oscillations, but the relationship between the oscillations in single cells and synchronized cultures is poorly understood. To understand this relationship and the coordination between metabolism and cell division, we collected and analyzed DNA-content, gene-expression and physiological data, at hundreds of time-points, from cultures metabolically-synchronized at different growth rates, carbon sources and biomass densities. The data enabled us to extend and generalize an ensemble-average-over-phases (EAP) model that connects the population-average gene-expression of asynchronous cultures to the gene-expression dynamics in the single-cells comprising the cultures. The extended model explains the carbon-source specific growth-rate responses of hundreds of genes. Our data demonstrate that for a given growth rate, the frequency of metabolic cycling in synchronized cultures increases with the biomass density. This observation underscores the difference between metabolic cycling in synchronized cultures and in single cells and suggests entraining of the single-cell cycle by a quorum-sensing mechanism. Constant levels of residual glucose during the metabolic cycling of synchronized cultures indicate that storage carbohydrates are required to fuel not only the G1/S transition of the division cycle but also the metabolic cycle. Despite the large variation in profiled conditions and in the time-scale of their dynamics, most genes preserve invariant dynamics of coordination with each other and with the rate of oxygen consumption. Similarly, the G1/S transition always occurs at the beginning, middle or end of the high oxygen consumption phases, analogous to observations in human and drosophila cells.Comment: 34 pages, 7 figure

Genetic Basis of Ammonium Toxicity Resistance in a Sake Strain of Yeast: A Mendelian Case.

High concentrations of ammonium at physiological concentrations of potassium are toxic for the standard laboratory strain of Saccharomyces cerevisiae In the original description of this metabolic phenotype, we focused on the standard laboratory strains of Saccharomyces In this study, we screened a large collection of S. cerevisiae natural isolates and identified one strain that is resistant to high concentrations of ammonium. This strain, K12, was isolated in sake breweries. When the K12 strain was crossed to the standard laboratory strain (FY4), the resulting tetrads displayed 2:2 segregation of the resistance phenotype, suggesting a single gene trait. Using a bulk segregant analysis strategy, we mapped this trait to a 150-kb region on chromosome X containing the TRK1 gene. This gene encodes a transporter required for high-affinity potassium transport in S. cerevisiae Data from reciprocal hemizygosity experiments with TRK1 deletion strains in K12 and BY backgrounds, as well as analysis of the deletion of this gene in the K12 strain, demonstrate that the K12 allele of TRK1 is responsible for ammonium toxicity resistance. Furthermore, we determined the minimal amount of potassium required for both the K12 and laboratory strain needed for growth. These results demonstrate that the gene encoded by the K12 allele of TRK1 has a greater affinity for potassium than the standard allele of TRK1 found in Saccharomyces strains. We hypothesize that this greater-affinity allele of the potassium transporter reduces the flux of ammonium into the yeast cells under conditions of ammonium toxicity. These findings further refine our understanding of ammonium toxicity in yeast and provide an example of using natural variation to understand cellular processes

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types

Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium

New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation ofammoniumconcentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells. Here we describe that the CLS shortening under amino acid restriction can be completely reverted by removing ammonium from the culture medium. Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions. When present in the culture medium,ammoniumimpaired the consumption of theauxotrophy-complementing amino acidsand caused in an improper cell cycle arrest of the culture.TOR1deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation ofammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.Julia Santos holds a Post-Doc fellowship (UMINHO/ BPD / 39/ 2013) funded by QREN-FEDER

Mapping of QTL for intermedium spike on barley chromosome 4H using EST-based markers

The lateral spikelets of two-rowed barley are reduced in size and sterile, but in six-rowed barley all three spikelets are fully fertile. The trait is largely controlled by alleles at the vrs1 locus on chromosome arm 2HL, as modified by the allele present at the I locus on chromosome arm 4HS. Molecular markers were developed to saturate the 4HS region by exploiting expressed sequence-tags, either previously mapped in barley to this region, or present in the syntenic region of rice chromosome 3. Collinearity between rice and barley was strong in the 4.8 cM interval BJ468164-AV933435 and the 10 cM interval AV942364-BJ455560. A major QTL for lateral spikelet fertility (the I locus) explained 44% of phenotypic variance, and was located in the interval CB873567-BJ473916. The genotyping of near-isogenic lines for I placed the locus in a region between CB873567 and EBmac635, and therefore the most likely position of the I locus was proximal to CB873567 in a 5.3 cM interval between CB873567-BJ473916

Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae

A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z_(3)EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z_(3)EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications

WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1-8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22-6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12-20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis

Pervasive Genetic Hitchhiking and Clonal Interference in 40 Evolving Yeast Populations

The dynamics of adaptation determines which mutations fix in a population, and hence how reproducible evolution will be. This is central to understanding the spectra of mutations recovered in evolution of antibiotic resistance1, the response of pathogens to immune selection2,3, and the dynamics of cancer progression4,5. In laboratory evolution experiments, demonstrably beneficial mutations are found repeatedly6–8, but are often accompanied by other mutations with no obvious benefit. Here we use whole-genome whole-population sequencing to examine the dynamics of genome sequence evolution at high temporal resolution in 40 replicate Saccharomyces cerevisiae populations growing in rich medium for 1,000 generations. We find pervasive genetic hitchhiking: multiple mutations arise and move synchronously through the population as mutational “cohorts.” Multiple clonal cohorts are often present simultaneously, competing with each other in the same population. Our results show that patterns of sequence evolution are driven by a balance between these chance effects of hitchhiking and interference, which increase stochastic variation in evolutionary outcomes, and the deterministic action of selection on individual mutations, which favors parallel evolutionary solutions in replicate populations

Cinderella; or Music and the Human Sciences. Unfootnoted Musings from the Margins

It has become fashionable among scholars to wax autobiographical with the reader, presumably to shed any remnant of the illusion (suggested implicitly by the conventional apparatus of a scholarly text and footnotes) that one might be speaking with an objective voice, or with an argument whose merits can be considered and even accepted without reference to personal and therefore circumstantial prejudice. Today's penchant for presumed full disclosure of one's subjective standpoint, however, is more likely either a species of authorial vanity masquerading as methodological scrupulousness or evidence of a greater interest in oneself than the subject one is writing about. In this case, the reader who wishes to distill the prejudices of the author and speculate on their origins must begin with the author's notion that one can talk effectively about the character and value of arguments by using procedures of reading and research that hold up under scrutiny and require no subjective apologetics. Botstein concludes that the definition of future methods of analysis, including the setting of the research agenda, cannot be undertaken from within the current traditions of music history or musicology