PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

PV技术的计算机处理及其在树木水分关系研究中的应用

ＰＶ技术的计算机处理及其在树木水分关系研究中的应用刘建伟,刘雅荣,王世绩（中国林业科学研究院林业研究所,北京１０００９１）ＣｏｍｐｕｔｅｒＰｒｏｃｅｓｓｉｎｇｏｆＰＶＣｕｒｖｅＡｎａｌｙｓｉｓａｎｄＩｔｓＡｐｐｌｉｃａｔｉｏｎｉｎｔｈｅＲｅｓｅａｒｃ...     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

河南木兰属9种植物过氧化物同工酶分析

本文采用聚丙烯酰胺凝胶电泳系统,测定了河南木兰属8种、1变种50多份的成熟叶片材料的过氧化物同工酶酶谱。测定结果表明,该属植物9种、变种的酶谱均有差异性,每种、变种均有特征酶谱,种间酶谱显著大于种内酶谱,根据其酶谱差异性,可以进行生物种、变种的鉴别和良种的选择。为免除其酶带Rf单一指标,鉴别生物种、变种可能出现的问题,作者创立了"酶谱多指标综合判断距离法"分析技术,首次将该属植物种、变种酶谱的酶带数目、Rf、酶带宽度及其活性强弱4个因子的差异性,进行编码联机、电脑运算分析,其结果与该属玉兰亚属内的玉兰派和辛夷派的形态分类相吻合。但是,从酶学观点不支持椭圆叶玉兰作为独立种的存在,以作为河南玉兰的变种为宜,也不支持河南玉兰派和腋花玉兰派的成立,以并入辛夷派为佳。     

湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

Resource availability at three presumed stages of a heathland succession on the Lüneburger Heide,Germany

Abstract. The availability of maj or plant resources was investigated in three vegetation types that were assumed to represent different stages of a secondary succession on heathland on the Lüneburger Heide, northwestern Germany. Canopy transmission and absorption of photosynthetically active radiation (PAR), soil-water availability, and nutrient (Ca, K, Mg, N, P) availability were monitored with high spatial and temporal resolution in (1) a Calluna vulgaris heathland, (2) a pioneer birch-pine forest and (3) a late-successional oak-beech forest, situated close to each other on comparable geological substrate (diluvial). Mean fractional transmission of PAR during summer decreased from 0.48 in the heathland to 0.04 in the oak-beech forest while the fractional canopy absorption increased from 0.49 to 0.92. Soil-water availability as indicated by the soil-water potential, was significantly influenced by differential canopy interception loss and characteristic rooting patterns in the three vegetation types. Annual mean nutrient concentrations in the equilibrium soil solution were similar or, for some elements, increased from the heathland to the birch-pine and the oak-beech forest despite a growing demand. A marked increase was found for the total nutrient pools in the soil-organic layer between early and late successional vegetation types. On the poor glacial parent material, nutrient pools seem to be strongly dependent on stand productivity and litter production which increased with succession. Thus, for nutrients, facilitation seems to be important in this type of succession.