Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Deltasnf2, and Deltaira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Deltapre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Deltasnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Deltasnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Deltasnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Deltasnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Deltasnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.     

Investigation of the impact of MIG1 and MIG2 on the physiology of Saccharomyces cerevisiae

The gene functions of MIG1 and MIG2 are well known for their role in glucose control in Saccharomyces cerevisiae. A prototrophic mig1 disruptant (T468) and mig1mig2 double disruptant (T475) as well as their congenic wild-type strain (CEN.PK 113-7D) were analysed for changes in their peripheral metabolism (batch cultivations on sugar mixtures) and central metabolism (batch and continuous cultivations as well as acceleratostats). Sucrose metabolism was alleviated of glucose control in the mig1 disruptant, and even more so in the mig1mig2 disruptant compared with their wild-type strain. The lag phase in a batch cultivation grown on a glucose-galactose mixture was reduced by 50% in either disruptant, i.e. additional disruption of MIG2 in a mig1 background did not further alleviate galactose metabolism from glucose control. In contrast, both disruptants exhibited a more stringent glucose control of maltose metabolism compared with the wild-type strain. Growing on glucose, the mig1mig2 double disruptant exhibited a 12% higher specific growth rate than the wild-type strain, as well as a significantly higher respiratory capacity.     

Signaling via the G protein alpha subunit FGA2 is necessary for pathogenesis in Fusarium oxysporum

Cloning and disruption of fga1, the gene encoding the G protein alpha subunit FGA1 in phytopathogenic fungus Fusarium oxysporum, has been reported previously, and the fga1 disruptants showed altered colony morphology, increased heat resistance, reduced conidiation and pathogenicity. To further evaluate the role of G protein signaling in this fungus, cloning of fga2, which encodes the second Galpha protein FGA2, was performed by PCR methods. The deduced primary structure of FGA2 (355 amino acid residues) showed high identity with other Galpha proteins, which belong to class III of fungal Galpha proteins. Disruption of fga2 led to higher heat resistance, similar to the fga1 disruptants, but pathogenicity was completely lost, unlike the fga1 disruptants. Alteration of colony morphology and conidiation, which was observed in the fga1 disruptants, was not observed in the fga2 disruptants. The fga1/fga2 double disruptants showed phenotypic alterations similar to the fga1 or fga2 single disruptants, but increase of heat resistance was much more pronounced than in each single disruptant.     

Dof基因家族调节植物生长发育功能的研究进展

Dof（DNA binding with one finger）蛋白是一类植物特异性转录因子,通常含有200～400个氨基酸和2个主要结构域。该家族成员的N 末端为高度保守的单锌指Dof结构域,具有与DNA和蛋白质相互作用的双重功能,其C末端的氨基酸序列则较为多变,是Dof蛋白重要的特异转录调控结构域。研究表明,Dof蛋白作为转录激活物或阻遏物参与了多方面的植物生长发育过程。随着基因组测序技术的发展,已有大量的Dof基因从植物基因组数据库中鉴定出来。该文对近年来国内外有关Dof基因家族的结构特点、全基因组鉴定、蛋白互作以及生物学功能等方面的研究进展进行综述,以期为Dof转录因子的深入研究提供参考。     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

植物Dof转录因子及其生物学功能

Dof(DNA binding with one finger)蛋白是植物特有的一类转录因子,包含一个C2-C2锌指,其N-末端保守的Dof结构域是既与DNA又和蛋白相互作用的双重功能域。在过去10多年的研究中,Dof蛋白在多种单子叶和双子叶植物中被分离。Dof蛋白作为转录的激活子或抑制子在植物的生长和发育中发挥重要作用。就Dof转录因子及其生物学功能的进展进行了综述。