Isolation and characterization of mutations affecting expression of the Δ9- fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae

AbstractExpression of the Δ9- fatty acid desaturase gene, OLE1, of Saccharomyces cerevisiae is negatively regulated transcriptionally and post-transcriptionally by unsaturated fatty acids. In order to isolate mutants exhibiting irregulation of OLE1 expression, we constructed an OLE1p–PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 promoter (OLE1p). By EMS mutagenesis, we isolated three classes of mutants, pfo1, pfo2 and pfo3 (positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (absence of oleic acid). Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the regulation of OLE1 expression at the transcriptional level while pfo2 mutants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step. In addition, four other classes of mutants, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that have mutations causing strong expression of the OLE1p–PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated. Results of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encoding global repressors. We also demonstrated that TUP1 and SSN6 gene products are required for full repression of OLE1 gene expression, by showing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript

Dust from Comet 209P/LINEAR during its 2014 Return: Parent Body of a New Meteor Shower, the May Camelopardalids

We report a new observation of the Jupiter-family comet 209P/LINEAR during its 2014 return. The comet is recognized as a dust source of a new meteor shower, the May Camelopardalids. 209P/LINEAR was apparently inactive at a heliocentric distance rh = 1.6 au and showed weak activity at rh < 1.4 au. We found an active region of <0.001% of the entire nuclear surface during the comet's dormant phase. An edge-on image suggests that particles up to 1 cm in size (with an uncertainty of factor 3-5) were ejected following a differential power-law size distribution with index q=-3.25+-0.10. We derived a mass loss rate of 2-10 kg/s during the active phase and a total mass of ~5x10^7 kg during the 2014 return. The ejection terminal velocity of millimeter- to centimeter-sized particles was 1-4 m/s, which is comparable to the escape velocity from the nucleus (1.4 m/s). These results imply that such large meteoric particles marginally escaped from the highly dormant comet nucleus via the gas drag force only within a few months of the perihelion passage.Comment: 18 pages, 4 figures, accepted on 2014 December 11 for publication in the Astrophysical Journal Letter

High-Throughput Cryopreservation of Plant Cell Cultures for Functional Genomics

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics

Metabolomics approach for determining growth-specific metabolites based on Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is the best MS technology for obtaining exact mass measurements owing to its great resolution and accuracy, and several outstanding FT-ICR/MS-based metabolomics approaches have been reported. A reliable annotation scheme is needed to deal with direct-infusion FT-ICR/MS metabolic profiling. Correlation analyses can help us not only uncover relations between the ions but also annotate the ions originated from identical metabolites (metabolite derivative ions). In the present study, we propose a procedure for metabolite annotation on direct-infusion FT-ICR/MS by taking into consideration the classification of metabolite-derived ions using correlation analyses. Integrated analysis based on information of isotope relations, fragmentation patterns by MS/MS analysis, co-occurring metabolites, and database searches (KNApSAcK and KEGG) can make it possible to annotate ions as metabolites and estimate cellular conditions based on metabolite composition. A total of 220 detected ions were classified into 174 metabolite derivative groups and 72 ions were assigned to candidate metabolites in the present work. Finally, metabolic profiling has been able to distinguish between the growth stages with the aid of PCA. The constructed model using PLS regression for OD600 values as a function of metabolic profiles is very useful for identifying to what degree the ions contribute to the growth stages. Ten phospholipids which largely influence the constructed model are highly abundant in the cells. Our analyses reveal that global modification of those phospholipids occurs as E. coli enters the stationary phase. Thus, the integrated approach involving correlation analyses, metabolic profiling, and database searching is efficient for high-throughput metabolomics

C[4]植物のナトリウム要求性に関する研究

京都大学0048新制・課程博士農学博士甲第4129号農博第561号新制||農||514(附属図書館)学位論文||S63||N1975(農学部図書室)UT51-89-A26京都大学大学院農学研究科農芸化学専攻(主査)教授 髙橋 英一, 教授 山田 康之, 教授 淺田 浩二学位規則第5条第1項該当Kyoto UniversityDFA