Isolation of Intact Chloroplasts from Dunaliella tertiolecta

Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO₂ was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O₂ evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O₂ and fixed 21 micromoles of CO₂ per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO₂ in air for 24 hours. On low CO₂ the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations.     

Desiccation Tolerance of Papaver dubium L. Pollen during Its Development in the Anther: Possible Role of Phospholipid Composition and Sucrose Content

Developing pollen of Papaver dubium L. becomes functional and desiccation tolerant at approximately 2 to 1 days prior to anthesis, coincident with degradation of starch and a doubling of the amount of sucrose, the primary soluble carbohydrate present. When anthers were taken from flower buds at 3 days before anthesis, pollen dehisced upon exposure to the ambient air. This dried pollen did not fluoresce with the vital stain fluorescein diacetate, had increased leakage of K⁺, and did not swell properly in a germination medium. In contrast, pollen became functional and desiccation resistant when these young anthers were first incubated in a water-saturated atmosphere for 30 hours. Phospholipid composition revealed no major differences over the last 3 days of development. When this immature pollen was liberated mechanically and allowed to mature in humid air, starch degraded and sucrose content nearly doubled, and the grains became largely functional and dehydration tolerant. Large unilamellar vesicles were prepared from isolated phospholipids to study dehydration-induced fusion and leakage. When dried in the presence of increasing concentrations of sucrose, vesicle integrity was progressively retained. These data indicate that pollen maturation during the last 3 days of development occurred independently from the parent plant. Sucrose may play an essential role in the acquired tolerance to severe dehydration.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

用改良的杆状病毒载体在家蚕中进行人α-干扰素基因的高水平表达

基因工程的目的之一是增加在细胞中所需的基因产物的表达量。首先用大量生产的宿主有大肠杆菌和一些其他的细菌。但是当细菌作为宿主时存在着几个问题,例如多肽糖基化缺陷、寄主蛋白酶造成的产物降解及缺乏分泌等。采用哺乳类动物细胞可排除上述问题,但要大量生产,仍然存在着许多与培养系统的复杂性及效率有关的问题。     

哈巴德布鲁克生态系统（H. B. E.）及其五号流域（W-5）研究

一、前言哈巴德布鲁克实验林(Hubbard Brook Experimental Forest)位于美国东北部新罕布什尔州中部的白山国家森林中。该地区位于典型温带湿润气候区内,年平均降水量为129.5cm,全年月平均降水量变化不大,冬雪夏雨。蒸发蒸腾量以每年6—9月为最大(Likens等,1977;Bormann等,1979)。该实验林为北美温带落叶阔叶林,属红果云杉(Picea rubens)-阔叶林。Hubbard Brook Ecosystem Study(HBES)是开始最     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: II. Illuminated Cells

The action spectrum for resetting the phase of the circadian clock in Chlamydomonas reinhardtii is different depending upon whether the light stimuli are presented to cells that were in darkness versus dim illumination before stimulation. In this report, we show that phase resetting of illuminated cells appears to be mediated by components of the photosynthetic apparatus. This conclusion is based upon the action spectrum for phase-shifting illuminated cells (which looks like that for photosynthesis) and upon the fact that inhibitors of photosynthetic electron transport also inhibit the light-induced phase shift of illuminated cells. Both of these characteristics differ from that of cells taken from darkness. We, therefore, believe that at least two resetting pathways for this circadian clock exist and that both of these pathways are ecologically significant.     

ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis

In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol.     

Regulation of sucrose-sucrose-fructosyltransferase in barley leaves

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.     

Two Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection

α-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two α-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the α-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both α-amylases produced in tobacco upon TMV infection.