We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding ␥-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.Ordinary commercial baker's yeast (mostly strains of Saccharomyces cerevisiae) is generally susceptible to damage during the freeze-thaw process and frozen storage and does not retain sufficient leavening ability after thawing (8,11). Freeze tolerance is a necessary characteristic of baker's yeast used in frozen doughs because postthaw leavening activity is essential prior to baking. It is known that the laboratory yeast S. cerevisiae induces trehalose synthesis during various stresses (1,10,34) and that induced trehalose functions as a stress protectant (7,16,23).We previously found that proline and charged amino acids, such as arginine and glutamate, have cryoprotective activities nearly equal to that of glycerol or trehalose in baker's yeast (26). It was shown previously that yeast cells with higher levels of proline are more tolerant of freezing than wild-type cells (13,14,22,26,28,30). Proline and trehalose were reported to preserve membrane structure and function during freezing (20). It has been suggested that proline can prevent ice nucleation and dehydration by forming strong hydrogen bonds with intracellular free water. ␥-Glutamyl kinase (GK; the PRO1 gene product) is the key enzyme in proline biosynthesis in S. cerevisiae, and GK activity is subjected to feedback inhibition by proline (22,32). Proline oxidase (PO; the PUT1 gene product) catalyzes the first step of the proline degradation pathway in S. cerevisiae (36). Interestingly, the D154N and I150T mutant GKs are less sensitive to proline feedback inhibition than the wild-type enzyme, and yeast cells expressing these mutated GKs accumulate proline and show higher tolerance for freezing than wild-type cells (22). With respect to industrial yeast, we previously constructed novel proline-accumulating sake yeast by disrupting the PUT1 gene and introducing the pro1(D154N) mutant allele and found that the resultant strain accumulated proline and was more tolerant of ethanol stress than the control strain (29). Furthermore, self-cloning (SC) diploid sake yeasts that accumulate proline have been developed, and their fermentation profiles during sake brewing have been analyzed (27). For the application of recombinant yeasts for commercial use, an SC yeast, which has no foreign genes or DNA sequences except for yeast DNA, may be more acceptable to consumers than a genetically modified (GM) yeast.Our objectives in this study were (i) to construct SC diploid baker's yeast strains that accumulate proline and (ii) to determine if intracellular proline enhances the freeze tolerance of baker's yeast strains in do...

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Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are environmentally persistent neurodevelopmental toxicants. The primary source of human exposure is the consumption of contaminated fish, seafood and marine mammals. However, little is known about the molecular mechanisms of MeHg and PCB toxicities and interactions between these contaminants. We investigated the functional profiles of differently expressed genes in the brains of offspring mice perinatally exposed to MeHg and/or PCBs to elucidate how these contaminants interact with each other. Pregnant mice (C57BL/6) were divided into four groups by exposure: (1) vehicle control, (2) MeHg alone, (3) PCBs alone, (4) MeHg + PCBs. Gene expression analysis of the brains of offspring mice was carried out with 4 x 44 K whole mouse genome's microarrays (Agilent) on postnatal day 1. The gene expression pattern of the MeHg exposure-group differed from that of the PCB-exposure group. The MeHg + PCB group expressed a larger number of genes, most of which were not expressed in the MeHg group or PCB group. It was revealed that gene expression was greatly increased, and the most altered genes were found with co-exposure. The genes were related to the functional categories of development, inflammation, calcium ion homeostasis, signal transduction, the ubiquitin-proteasome pathway and detoxication. The ubiquitin-proteasome system and detoxication categories might function for protection against the toxicity induced by co-exposure to MeHg and PCBs. These results suggest that co-exposure does not simply exacerbate the toxicity of MeHg alone or PCB alone, but stimulates a protection system.

The effects of 17β-estradiol (E2) were evaluated using the medaka DNA microarray representing 36,398 genes. We first evaluated chronic effects on medaka exposed to E2 at different concentrations for 60 days posthatch. At ≥ 30 ng/L of E2 severe reproductive impairments such as sex reversal were observed. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were then exposed to E2 at various concentrations (3, 30, 100 ng/L) for up to 7 days. Microarray analyses of the E2-exposed larvae revealed that exposure to E2 up-regulated and down-regulated 339 and 105 genes, respectively. The up-regulated genes included ones involved in the p53 signaling pathway, apoptosis, and growth and development, in addition to well-known biomarkers such as vitellogenin and choriogenins. Down-regulated genes included heat shock proteins and estrogen receptors. Most of the up-regulated genes encoding the p53 signaling pathway, apoptosis, and growth and development exhibited a dose-dependent increase in gene expression, whereas the down-regulated genes in the heat shock protein category showed a dose-dependent decrease in gene expression. Time course experiments suggested that the E2 treatment attenuated the time-dependent changes in gene expressions of these genes. Among the genes related to oocyte maturation, estrogen-regulated genes such as choriogenins and vitellogenins were dramatically induced in response to E2 exposure, whereas other steroid-regulated genes such as zona pellucida-domain proteins did not change in gene expression by the E2 treatment. Results suggest that transcriptomic studies on larval medaka help elucidate the effects caused by endocrine disruptors on various biological pathways in vertebrate development.

In the modern baking industry, high-sucrose-tolerant (HS) and maltose-utilizing (LS) yeast were developed using breeding techniques and are now used commercially. Sugar utilization and high-sucrose tolerance differ significantly between HS and LS yeasts. We analysed the gene expression profiles of HS and LS yeasts under different sucrose conditions in order to determine their basic physiology. Twoway hierarchical clustering was performed to obtain the overall patterns of gene expression. The clustering clearly showed that the gene expression patterns of LS yeast differed from those of HS yeast. Quality threshold clustering was used to identify the gene clusters containing upregulated genes (cluster 1) and downregulated genes (cluster 2) under high-sucrose conditions. Clusters 1 and 2 contained numerous genes involved in carbon and nitrogen metabolism, respectively. The expression level of the genes involved in the metabolism of glycerol and trehalose, which are known to be osmoprotectants, in LS yeast was higher than that in HS yeast under sucrose concentrations of 5-40%. No clear correlation was found between the expression level of the genes involved in the biosynthesis of the osmoprotectants and the intracellular contents of the osmoprotectants. The present gene expression data were compared with data previously reported in a comprehensive analysis of a gene deletion strain collection. Welch's t-test for this comparison showed that the relative growth rates of the deletion strains whose deletion occurred in genes belonging to cluster 1 were significantly higher than the average growth rates of all deletion strains.