Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.     

Homology-Based Interactions between Small RNAs and Their Targets Control Dominance Hierarchy of Male Determinant Alleles of Self-Incompatibility in Arabidopsis lyrata

Self-incompatibility (SI) is conserved among members of the Brassicaceae plant family. This trait is controlled epigenetically by the dominance hierarchy of the male determinant alleles. We previously demonstrated that a single small RNA (sRNA) gene is sufficient to control the linear dominance hierarchy in Brassica rapa and proposed a model in which a homology-based interaction between sRNAs and target sites controls the complicated dominance hierarchy of male SI determinants. In Arabidopsis halleri, male dominance hierarchy is reported to have arisen from multiple networks of sRNA target gains and losses. Despite these findings, it remains unknown whether the molecular mechanism underlying the dominance hierarchy is conserved among Brassicaceae. Here, we identified sRNAs and their target sites that can explain the linear dominance hierarchy of Arabidopsis lyrata, a species closely related to A. halleri. We tested the model that we established in Brassica to explain the linear dominance hierarchy in A. lyrata. Our results suggest that the dominance hierarchy of A. lyrata is also controlled by a homology-based interaction between sRNAs and their targets.     

Fluorescent whitening agents in Tokyo Bay and adjacent rivers: their application as anthropogenic molecular markers in coastal environments

Two kinds of stilbene-type fluorescent whitening agents (i.e., DSBP and DAS1), minor components of laundry detergents, were analyzed in surface waters of Tokyo Bay and adjacent rivers and in sewage effluents to examine their usefulness as molecular markers in the marine environment. Sensitive determination using HPLC (high performance liquid chromatography) with fluorescence detection with postcolumn UV radiator was employed. DSBP and DAS1 were found in Tokyo rivers at concentrations of a few microg/L and approximately 1 microg/L, respectively. DSBP and DAS1 were widely distributed in Tokyo Bay waters at concentrations in the range of 0.019-0.264 microg/L and 0.021-0.127 microg/L, respectively. Comparison of these concentrations with those in sewage effluents (DSBP: 8 microg/L and DAS1: 2.5 microg/L on average) yielded sewage dilutions in Tokyo Bay on the order of 10(2). FWAs-salinity diagram in the Tamagawa Estuary showed fairly conservative behaviors of the FWAs with approximately 20% and approximately 10% removal of DSBP and DAS1, respectively. This is thought to be caused by photodegradation. The persistent nature of FWAs and their widespread distribution in coastal environments demonstrates the utility of FWAs in tracing the behavior of water from rivers and sewage outfalls. The DSBP/DAS1 ratio showed a decreasing trend from sewage effluents, to rivers, to Tokyo Bay, indicating selective photodegradation of DSBP. The DSBP/DAS1 ratio is proposed as an index of the degree of photodegradation and residence time and freshness of water mass in coastal environments.     

Characterization and CO2 sorption properties of poly[methacryloxypropylheptacyclopentyl‐T8‐silsesquioxane‐ co‐3‐methacryloxypropyltris(trimethylsiloxy)silane]

Poly[methacryloxypropylheptacyclopentyl‐T8‐silsesquioxane (MAPOSS)‐co‐3‐methacryloxypropyltris(trimethylsiloxy)silane (SiMA)] was synthesized through free radical polymerization. The physical and carbon dioxide (CO₂) sorption properties of the copolymer membranes were investigated in terms of the MAPOSS content. As the MAPOSS content increases, the membrane density increased, suggesting a decrease in the fractional free volume. In addition, the thermal stability was improved with increasing the MAPOSS content. These are because of the polyhedraloligomericilsesquioxane (POSS) units that restrict the high mobility of poly(SiMA) segments. The glass transition temperature, T_g of the copolymers was single T_g based on the differential scanning calorimetry, suggesting that the copolymers were random and not phase separation. Based on the CO₂ sorption measurement, the POSS units play a role in reducing Henry's dissolution by suppressing the mobility of the poly(SiMA) component, while POSS units increase the nonequilibrium excess free volume, which contributes to the Langmuir dissolution. Based on these results, the introduction of MAPOSS unit is one of the effective ways to improved the thermal stability and CO₂ sorption property due to the enhancement of the polymer rigidity. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

URINARY 2-HYDROXYFLUORENE AND 1-HYDROXYPYRENE LEVELS IN SMOKERS AND NONSMOKERS IN JAPAN AND THAILAND

2-Hydroxyfluorene (2-OHF) and 1-hydroxypyrene (1-OHP) in urine samples of smokers and nonsmokers were determined separately by using two high-performance liquid chromatography (HPLC) systems with fluorescence detection. Urine samples were collected from the subjects who lived in Japan and Thailand, and were not occupationally exposed to polycyclic aromatic hydrocarbons (PAHs). The mean concentrations of 2-OHF and 1-OHP of Thai smokers (0.75 and 3.03 μmol/mol creatinine) and nonsmokers (0.22 and 0.91 μmol/mol creatinine) were both higher than those of Japanese smokers (0.26 and 0.12 μmol/mol creatinine) and nonsmokers (0.04 and 0.06 μmol/mol creatinine), respectively. The difference between smokers and nonsmokers was more significant for 2-OHF than for 1-OHP, reflecting the higher intake of fluorene in the vapor phase by the smoking. Moreover, the higher urinary levels of both 2-OHF and 1-OHP were observed in Thai nonsmokers than those in Japanese nonsmokers, suggesting the higher background exposure to PAHs of Thai subjects.     

Transgenic melon (cucumis melo L.) and potential for expression of novel proteins important to food industry

Abstract

Melon (Cucumis melo L.) is an important fruit crop cultivated widely in every region of the world. Our laboratory is targeting this species for production of novel proteins important to food industry. Prior to expression of protein of interest in transgenic melon an efficient genetic transformation system has to be developed. In this context we are testing a wide variety of promoters fused to reporter gene for β‐glucuronidase (GUS) for expression specifically in melon fruits. In this study in melon, salicylic acid‐inducible promoter region of pathogenesis‐related protein gene (PR1a) of tobacco fused to β‐glucuronidase (GUS) gene was introduced into melon via Agrobacterium‐mediated gene transfer using a binary vector system. Gene transfer was effective when Agrobacterium virulence factors like acetosyringone (100 μM) and low pH (5.2) were provided during the co‐culture step. Transformed shoots were recovered from benzyladenine‐induced cut cotyledons using kanamycin gene as a selective marker. Regeneration of shoots from cotyledons was stimulated by providing 10 mM proline in the shoot organogenesis medium. Southern and Northern blot analysis of transformants confirmed the presence of β‐glucuronidase gene in two selected clones J‐3 and PR‐G. The transformants also showed high β‐glucuronidase activity after salicylic acid treatment. Thiamine, a previously known inducer of pathogenesis‐related protein, stimulated β‐glucuronidase in J‐3 but not PR‐G melon transformants tested in this study. These studies showed that tobacco PR1a promoter region can be expressed in melon and it was stimulated by salicylic acid. This indicates the potential to use the promoter region of tobacco PR1a for genetic improvement of melon for specific food processing‐related characteristics or for expression of novel food‐related proteins. The promoter region could be used to drive specific target genes under stress or salicylic acid induced conditions.     

Crystallographic, Thermoelectric, and Mechanical Properties of Polycrystalline Ba8Al x Si46−x Clathrates

The Al content dependence of crystallographic, thermoelectric, and mechanical properties is reported for polycrystalline Ba₈Al _x Si_46?x (nominal x = 15 to 17) clathrates prepared by combining arc melting and spark plasma sintering methods. The elastic constants and the coefficient of thermal expansion (CTE), which are also important properties for designing thermoelectric devices, are presented. Powder x-ray diffraction, scanning electron microscopy, and energy-dispersive x-ray spectroscopy (EDX) indicate that the type I clathrate is the major phase of the samples but impurity phases (mainly BaAl₂Si₂, Si, and Al) are included in the samples with high Al contents. The actual Al content x determined by EDX ranges from approximately 14 to 15. The absolute value of the Seebeck coefficient increases and the electrical conductivity decreases as the Al content increases. The changes in Seebeck coefficient and electrical conductivity are explained in terms of the dependence of the carrier concentration on the Al content. The elastic constants and the CTE of the samples depend weakly on the Al content. Some of the properties are compared with reported data of single crystals of Ba₈Al₁₆Ge₃₀, Ba₈Ga₁₆Ge₃₀, Sr₈Ga₁₆Ge₃₀, silicon, and germanium as standard references. The effective mass, Hall carrier mobility, and lattice thermal conductivity, which govern the transport properties, are determined to be ~ 2.4m ₀, ~ 7 cm² V^?1 s^?1, and ~ 1.3 W m^?1 K^?1, respectively, for actual Al content x of about 14.77. The thermoelectric figure of merit ZT is estimated to be about 0.35 at 900 K for actual Al content x of about 14.77.     

Production technology of large-area multicrystalline silicon solar cells

In 1996 a conversion efficiency of 17.1% had been obtained on 15 cm×15 cm mc-Si solar cell. In this paper, large-scale production technology of the high-efficiency processing will be discussed. Enlarging reactive ion etching (RIE) equipment size, technology of passivation, and fine contact grid with low resistance by screenprinted metallization, which is firing through PECVD SiN, have been investigated.     

Synthesis of geranyl acetate by lipase entrap-immobilized in cellulose acetate-TiO2 gel fiber

Lipase from Candida antarctica was entrap-immobilized in cellulose acetate-TiO₂ gel fiber (fiber-immobilized lipase) by the sol-gel method. Syntheses of geranyl acetate and citronellyl acetate catalyzed by the fiber-immobilized lipase were studied in heptane solution. Conversions reached 85% for geranyl acetate after 100 h, and 75% for citronellyl acetate after 80 h, and these values were almost identical to those for syntheses catalyzed by nonimmobilized lipase, although the reaction rate was decreased by immobilization. Compared to those of the non-immobilized lipase and commercially available immobilized lipase (Novozyme 435), the activity of the fiber-immobilized lipase was not particularly affected by changes in reaction conditions, such as bulk water content or substrate concentration. The fiber-immobilized lipase retained a high level of activity after six repeated uses, and almost no enzyme leakage from fiber was observed. However, the reactivity of the fiber-immobilized lipase was depressed at higher temperature, presumably due to dehydration by thermal contraction of the gel fiber.