Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Effect of polyglycerol esters of fatty acids on physicochemical properties and stability of β‐carotene nanodispersions prepared by emulsification/evaporation method

The effects of six different polyglycerol esters of fatty acids (PGEs) as nonionic emulsifiers on the physicochemical properties and stability of β‐carotene nanoparticles in oil‐in‐water dispersions produced by an emulsification/evaporation technique were examined. The β‐carotene particle size was measured by a laser diffraction technique, and the stability and retention of β‐carotene during various preparation steps and storage were determined by HPLC. In the prepared nanodispersions the β‐carotene particle size decreased with increasing degree of glycerol polymerisation and decreasing carbon number of the fatty acid group in the PGE. The particle size of β‐carotene in nanodispersions containing polyglycerol monooleate was generally larger than that in the presence of polyglycerol monolaurate. During storage at 4 °C, although the β‐carotene content in the nanodispersions showed a significant ( P < 0.05) decrease with increasing storage period, the size distribution of β‐carotene was almost unchanged in all prepared nanodispersions. In general, the mean diameter of β‐carotene nanoparticles ranged from 85 to 132 nm. In the light of their ability to physically stabilise β‐carotene particle formation, it is suggested that PGEs with a high degree of glycerol polymerisation may be useful in the preparation of β‐carotene nanodispersions. The best stabilisation was obtained using 10 g kg^?1 decaglycerol monolaurate. Copyright © 2004 Society of Chemical Industry     

The influence of hydrochloric acid on the effect of caproic acid in preventing aerobic deterioration of silages

In five experiments of the same design using silages made from wilted Italian ryegrass (dry matter 22.7–40.9%), the effects of caproic acid (C₆) (10 mmol kg^?1) with and without 6_N-HCl (20 ml kg^?1), applied at ensiling or at opening, in preventing aerobic deterioration of silages after opening silos were investigated. Application of C₆ either at ensiling or at opening was partially effective in preventing temperature increase, pH rise and breakdown of organic acids during the 7-day aerobic exposure period after opening the silos. Addition of HCl reduced the pH of silages irrespective of the time of application. However, no improvement of the effect of C₆ due to HCl supplementation was observed as far as the prevention of the aerobic deterioration was concerned. Treatment with C₆+ HCl at ensiling inhibited silage fermentation more extensively than C₆ alone. Viable counts of yeasts and moulds in the silages treated with C₆ or C₆+ HCl at ensiling tended to be lower than those in the control silages. Considerable increases in the counts of moulds were found in the control silages during the aerobic exposure period.     

Aspects of gasification and structure in cokes from coals

Cokes were prepared from nine coals of different rank and characterized by surface area measurement, reactivity to carbon dioxide at 1473K and Raman-laser spectroscopy. Rates of gasification of cokes on a unit surlface area basis (K¹ = g m^?2 min^?1) decreased with increasing rank of parent coal based on maximum oil reflectances. However rates of gasification could not be related to coke structure as measured by Raman-laser spectroscopy.     

Influence of different feeding systems on the growth performance and muscle development of Japanese Black steers

This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu) on antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells

We found that two distinct flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu), isoquercitrin (Isq) and hyperin (Hyp), are capable of inhibiting antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells. In order to elucidate the underlying mechanisms, we examined effects of Isq and Hyp on cellular responses induced by antigen stimulation. Treatment with both Isq and Hyp markedly inhibited antigen-stimulated elevation of intracellular free Ca²⁺ concentration and reactive oxygen species (ROS). Isq and Hyp did not affect NADPH oxidase (NOX) activity, but they possessed DPPH radical-scavenging activity similar to that of epigallocatechin gallate, a potent anti-oxidant, Finally, Isq and Hyp showed little or no effects on Ag-stimulated Syk activation or phosphorylation of signalling molecules. These results indicate that inhibition of antigen-stimulated degranulation by Isq and Hyp is mainly due to suppression of intracellular Ca²⁺ elevation, which is caused by direct scavenging of ROS that are generated by NOX. Our findings suggest that Isq and Hyp, isolated from the peel of persimmon, would be beneficial for alleviating symptoms of type I allergy.     

Occurrence,horizontal transfer and degeneration of VDE intein family in Saccharomycete yeasts

VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae. There have been two independent subfamilies of VDE, one from S. cerevisiae strain X2180-1A and the other from Saccharomyces sp. DH1-1A in the host VMA1 gene, and they share the identity of 96.3%. In order to search the occurrence, intra/interspecies transfer and molecular degeneration of VDE, complete sequences of VMA1 in 10 strains of S. cerevisiae, eight species of saccharomycete yeasts, Candida glabrata and Kluyveromyces lactis were determined. We found that six of 10 S. cerevisiae strains contain VDEs 99.7-100% identical to that of the strain X2180-1A, one has no VDE, whereas the other three harbour VDEs 100% identical to that of the strain DH1-1A. S. carlsbergensis has two VMA1s, one being 99.8% identical to that of the strain X2180-1A with VDE 100% identical to that of the strain DH1-1A and the other containing the same VMA1 in S. pastorianus with no VDE. This and other evidence indicates that intra/interspecies transmissions of VDEs have occurred among saccharomycete yeasts. Phylogenetic analyses of VMA1 and VDE suggest that the S. cerevisiae VDEs had branched earlier than other VDEs from an ancestral VDE and had invaded into the host loci as relatively late events. The two VDEs seemed to degenerate in individual host loci, retaining their splicing capacity intact. The degeneration of the endonuclease domains was distinct and, if compared, its apparent rate was much faster than that of the protein-splicing domains.     

Saccharomyces sensu stricto: systematics, genetic diversity and evolution

Saccharomyces sensu stricto is a species complex that includes most of the yeast strains relevant in the fermentation industry as well as in basic science. The taxonomy of these yeasts has always been controversial, particularly at species level. Over the years, the grouping of Saccharomyces sensu stricto yeasts has undergone changes in accordance with the system employed in classifying yeast cultures. Names of species and single isolates have also undergone changes that have caused confusion for yeast scientists and fermentation technologists. Recent findings have demonstrated that Saccharomyces hayanus and S. pastorianus are not homogeneous and do not seem to be natural groups. The present work examines the current trends in systematics studies, evidences the importance and mechanism of genetic variation and explores the most recent evolutionary theories as a way to elucidate the mechanism of speciation and achieve a more natural grouping of Saccharomyces sensu stricto species.