Is there replication-associated mutational pressure in the Saccharomyces cerevisiae genome?

Compositional bias of yeast chromosomes was analysed using detrended DNA walks. Unlike eubacterial chromosomes, the yeast chromosomes did not show the specific asymmetry correlated with origin and terminus of replication. It is probably a result of a relative excess of autonomously replicating sequences (ARS) and of random choice of these sequences in each replication cycle. Nevertheless, the last ARS from both ends of chromosomes are responsible for unidirectional replication of subtelomeric sequences with pre-established leading/lagging roles of DNA strands. In these sequences a specific asymmetry is observed, resembling the asymmetry introduced by replication-associated mutational pressure into eubacterial chromosomes.     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho^?) alleles of STE4: ste4-T320?A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

Influence of culture conditions on the production of heterologous interleukin 1β by Kluyveromyces lactis

Summary Under the control of the repressible PHO5 promoter, the expression of gene encoding interleukin 1 (Il1) was derepressed when the medium was depleted of free inorganic phosphate (Pi). Maximum heterologous protein synthesis was obtained in the presence of 75 mg KH₂PO₄/1 (for 20 g glucose/l). The successful heterologous protein production greatly depends on nutritional culture conditions as Il1 production efficiency was increased by 83% through optimization of the growth medium. Comparison of different phosphate-limited cultivation strategies led to the development of a batch culture procedure with nutrient pulses to delay induced oxido-fermentative glucose metabolism and increase the Il1 production to 135 mg/l.     

The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.     

Why does the brain (not) have glycogen?

In the present paper we formulate the hypothesis that brain glycogen is a critical determinant in the modulation of carbohydrate supply at the cellular level. Specifically, we propose that mobilization of astrocytic glycogen after an increase in AMP levels during enhanced neuronal activity controls the concentration of glucose phosphates in astrocytes. This would result in modulation of glucose phosphorylation by hexokinase and upstream cell glucose uptake. This mechanism would favor glucose channeling to activated neurons, supplementing the already rich neuron-astrocyte metabolic and functional partnership with important implications for the energy compounds used to sustain neuronal activity. The hypothesis is based on recent modeling evidence suggesting that rapid glycogen breakdown can profoundly alter the short-term kinetics of glucose delivery to neurons and astrocytes. It is also based on review of the literature relevant to glycogen metabolism during physiological brain activity, with an emphasis on the metabolic pathways identifying both the origin and the fate of this glucose reserve.     

What is the function of nitrogen catabolite repression in Saccharomyces cerevisiae?

In contrast to the previously held notion that nitrogen catabolite repression is primarily responsible for the ability of yeast cells to use good nitrogen sources in preference to poor ones, we demonstrate that this ability is probably the result of other control mechanisms, such as metabolite compartmentation. We suggest that nitrogen repression is functionally a long-term adaptation to changes in the nutritional environment of yeast cells.     

What properties characterize the hub proteins of the protein-protein interaction network of Saccharomyces cerevisiae?

Background  

Most proteins interact with only a few other proteins while a small number of proteins (hubs) have many interaction partners. Hub proteins and non-hub proteins differ in several respects; however, understanding is not complete about what properties characterize the hubs and set them apart from proteins of low connectivity. Therefore, we have investigated what differentiates hubs from non-hubs and static hubs (party hubs) from dynamic hubs (date hubs) in the protein-protein interaction network of Saccharomyces cerevisiae.     

Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes

Applied Microbiology and Biotechnology - It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this...     

Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide

Flow cytometric monitoring of propidium iodide (PI) uptake is a well-established and rapid method for monitoring cell death and is used on the basis that the intact membrane of viable cells excludes the propidium ion and that loss of this permeability barrier represents irreparable damage and thus cell death. These assumptions are typically based on analysis of live and killed cells. Here we have identified stress levels that lead to a loss of viability of a proportion of Saccharomyces cerevisiae cells and under these conditions we show that there is a subpopulation of cells that can take up PI during and immediately following exposure to stress but that a short incubation allows repair of the membrane damage such that subsequent exposure to PI does not result in staining. Irrespective of the stress applied, approximately 7% of cells exhibited the ability to repair. These results indicate that the level of damage that the yeast cell membrane can sustain and yet retain the ability to repair is greater than previously recognized and care must therefore be taken in using the terms 'PI-positive' and 'dead' synonymously. We discuss these findings in the context of the potential for such environmental stress-induced, transient membrane permeability to have evolutionary implications via the facilitation of horizontal gene transfer.     

How much does the amphioxus genome represent the ancestor of chordates?

One of the main motivations to study amphioxus is its potential for understanding the last common ancestor of chordates, which notably gave rise to the vertebrates. An important feature in this respect is the slow evolutionary rate that seems to have characterized the cephalochordate lineage, making amphioxus an interesting proxy for the chordate ancestor, as well as a key lineage to include in comparative studies. Whereas slow evolution was first noticed at the phenotypic level, it has also been described at the genomic level. Here, we examine whether the amphioxus genome is indeed a good proxy for the genome of the chordate ancestor, with a focus on protein-coding genes. We investigate genome features, such as synteny, gene duplication and gene loss, and contrast the amphioxus genome with those of other deuterostomes that are used in comparative studies, such as Ciona, Oikopleura and urchin.     

Does Aqueous Fullerene Inhibit the Growth of Saccharomyces cerevisiae or Escherichia coli?

Studies reporting on potentially toxic interactions between aqueous fullerene nanoparticles (nC(60)) and microorganisms have been contradictory. When known confounding factors were avoided, growth yields of Saccharomyces cerevisiae and Escherichia coli cultured in the presence and absence of independently prepared lots of underivatized nC(60) were found not to be significantly different.     

Do disparate mechanisms of duplication add similar genes to the genome?

Gene duplication is the fundamental source of new genes. Biases in duplication have profound implications for the dynamics of gene content during evolution. In this article, we compare genes arising from whole gene duplication (WGD), smaller scale duplication (SSD) and singletons in Saccharomyces cerevisiae. Our results demonstrate that genes duplicated by WGD and SSD are similarly biased with respect to codon bias and evolutionary rate, although differing significantly in their functional constituency.     

Why homocysteine-lowering therapy does not have beneficial effects on patients with cardiovascular disease?

Homocysteine is a sulfur-containing amino acid produced during the metabolism of methionine and elevated plasma levels of homocysteine have been linked to an increased risk of atherosclerosis and cardiovascular ischemic events by numerous authors. Several mechanisms by which elevated homocysteine impairs vascular function have been proposed including impairment of endothelial function and at least some of those mechanisms are induced via homocysteine-associated DNA hypomethylation. Oral administration of folic acid and B vitamins, required for remethylation of homocysteine to methionine, decreased plasma total homocysteine levels but clinical trials using folic acid and B vitamins did not confirm that the decreased plasma levels of homocysteine through diet or drugs may be paralleled by a reduction in cardiovascular risk. In our view a plausible explanation for the discordance between the epidemiologic studies and the results of the clinical trials may be related to the homocysteine-associated global DNA hypomethylation which cannot easily be reversed by homocysteine-lowering therapy.     

Inbreeding at the edge: does inbreeding depression increase under more stressful conditions?

Edge populations are frequently small and subject to stressful conditions that may compromise their long‐term viability. Inbreeding can play an important role in small populations by reducing genetic diversity, leading to the fixation of deleterious mutations and, finally, carrying populations to an extinction vortex through inbreeding depression. Although stressful conditions may enhance the intensity of inbreeding depression, evidence to date is inconclusive in marginal habitats. Local adaptation, promoting native genotypes, and gene flow, reducing allele fixation, are two factors that can have different effects on the intensity of inbreeding depression. Three populations of Silene ciliata distributed across an elevation gradient at the southernmost edge of the species distribution were used for this study. Several fitness components – germination, survival and growth rate – were compared between inbred seedlings and seedlings from within‐ and between‐population outcrosses, both in the field and controlled conditions. Overall, inbred seedlings had lower fitness than outcrossed seedlings. For most of the variables analysed, similar inbreeding depression effects were found in all three populations, but, for seed weight and seedling survival curve, inbreeding depression was only found in the low altitude population. Similarly, inbreeding depression was more evident in the field than in controlled chamber conditions. Outcrosses between populations contributed to an increase in most fitness estimates and populations, suggesting that the benefits of reducing inbreeding depression overrode the potentially deleterious effects of disrupting local adaptation. Our results suggest that inbreeding depression plays an important role in the fitness of early life stages of Silene ciliata at its southernmost distribution limit, but only provided partial support to the hypothesis that stressful conditions enhance the expression of inbreeding depression.     

Why does oriental arborvitae grow better when mixed with black locust: Insight on nutrient cycling?

To identify why tree growth differs by afforestation type is a matter of prime concern in forestry. A study was conducted to determine why oriental arborvitae (Platycladus orientalis) grows better in the presence of black locust (Robinia pseudoacacia) than in monoculture. Different types of stands (i.e., monocultures and mixture of black locust and oriental arborvitae, and native grassland as a control) were selected in the Loess Plateau, China. The height and diameter at breast height of each tree species were measured, and soil, shoot, and root samples were sampled. The arbuscular mycorrhizal (AM) attributes, shoot and root nutrient status, height and diameter of black locust were not influenced by the presence of oriental arborvitae. For oriental arborvitae, however, growing in mixture increased height and diameter and reduced shoot Mn, Ca, and Mg contents, AM fungal spore density, and colonization rate. Major changes in soil properties also occurred, primarily in soil water, NO₃‐N, and available K levels and in soil enzyme activity. The increase in soil water, N, and K availability in the presence of black locust stimulated oriental arborvitae growth, and black locust in the mixed stand seems to suppress the development of AM symbiosis in oriental arborvitae roots, especially the production of AM fungal spores and vesicles, through improving soil water and N levels, thus freeing up carbon to fuel plant growth. Overall, the presence of black locust favored oriental arborvitae growth directly by improving soil water and fertility and indirectly by repressing AM symbiosis in oriental arborvitae roots.     

Important Role of Catalase in the Production of ��-carotene by Recombinant Saccharomyces cerevisiae under H2O2 Stress

The effect of H(2)O(2) supplement on cell growth and β-carotene productions in recombinant Saccharomyces cerevisiae CFW-01 and CFW-01 ctt1 deficiency in cytosolic catalase were investigated in shaking flasks. The results showed that supplement of H(2)O(2) (0.5 and 1.0 mM) can significantly stimulate the β-carotene production. However, β-carotene levels of CFW-01 ctt1Δ under 0.5 and 1 mM H(2)O(2) were 16.7 and 36.7% lower than those of CFW-01, respectively. Although lacking cytosolic catalase, no significant differences in cell growth were observed between CFW-01 ctt1Δ and CFW-01 under the same level of H(2)O(2) stress. These results suggest that β-carotene can act as an antioxidant to protect the recombinant yeast from H(2)O(2) oxidative damage in the absence of cytosolic catalase. However, catalase still plays an important role in the production of β-carotene under H(2)O(2) stress. If catalase can not timely decompose H(2)O(2), the free radicals such as OH· derived from H(2)O(2) can result in decrease of β-carotene concentration. Therefore, in the production of β-carotene by H(2)O(2) stress, not only the level of oxidative stress, but also the activities of catalase in cells should be considered.     

Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.