首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
For the first time, the 31P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 106 vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H+ exchanges at the tonoplast. The 31P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.  相似文献   

2.
Sperm cells were isolated from corn (Zea mays L.) tricellular pollen grains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 × 106 per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.  相似文献   

3.
Useful Host-Vector Systems in Bacillus stearothermophilus   总被引:4,自引:2,他引:2       下载免费PDF全文
We isolated a highly transformable thermophile, Bacillus stearothermophilus SIC1, which exhibited the following features. The growth temperature ranged from 45 to 65°C in L broth. The maximum cell concentration in 2L broth (2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2) was determined as an optical density at 660 nm of 7.8, and the generation time was 11 min at 60°C. Strain SIC1 was a prototroph and was transformed by the protoplast procedure not only with repB plasmids (high-copy-number plasmids such as pTB913 and pUB110) but also with repA plasmids (low-copy-number plasmids such as pTB53). Transformation efficiencies with repB and repA plasmids were about 2 × 106 to 5 × 106 and 5 × 104 transformants per μg of DNA, respectively. The transformant carrying plasmid pTB913Y/K could grow at 63°C in the presence of kanamycin. The regeneration frequency of protoplasts was 60%, and only 1 day was needed for regeneration at 55°C.  相似文献   

4.
Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 106 electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10−4. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.  相似文献   

5.
We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 × 105 protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.  相似文献   

6.
Chlorine Demand and Inactivation of Fungal Propagules   总被引:2,自引:2,他引:0       下载免费PDF全文
Conidia of filamentous fungi, vegetative yeast cells, and coliform bacteria were tested to determine their chlorine demand and their sensitivity to chlorine inactivation. Levels of chlorine demand for the various conidia, yeast, and coliforms were, respectively, 3.6 × 10−9 to 3.2 × 10−8, 1.2 × 10−9 to 8.0 × 10−9, and 2.5 × 10−11 to 6.3 × 10−10 mg of chlorine per propagule. Preliminary evidence suggests that the chlorine demand per propagule increases as the number of propagules per milliliter decreases. In general, conidia showed greatest resistance to chlorine inactiviation, followed by the yeast and coliforms. Inactivation by chlorine was influenced by pH, with inactivation (chlorine activity) falling in the order pH 5 > 7 > 8.  相似文献   

7.
Cultures of coliform and Proteus bacteria, mostly from foods, were tested for their effect on growth of Staphylococcus aureus in Trypticase Soy Broth. Inhibition of the staphylococcus by these competitors increased with increasing proportions of inhibiting (effector) bacteria in the inoculum and decreasing incubation temperatures (37 to 10 C). Time required for 2 × 104 staphylococci to increase to 5 × 106 cells per milliliter, the minimal number assumed to be necessary for food poisoning, varied with the species of effector, the original ratio of effector bacteria to staphylococci in the medium, and the incubation temperature. When the original ratio was 100:1, the staphylococci did not reach 5 × 106 cells per milliliter at 10, 15, 22, or 30 C (with one exception), when growing with cultures representing six species of coliform bacteria and two of Proteus. When the ratio was 1:1, all effectors either greatly delayed the staphylococcus or prevented it from reaching hazardous numbers at 15 C, six of the eight caused a delay of 2 to 3 hr at 22 C, and only Escherichia coli delayed the coccus at 30 C. All effectors were ineffective at 22 and 30 C when original numbers of effectors and staphylococci were in the ratio 1:100. Greatest overall inhibition was by E. coli, E. freundii, and Proteus vulgaris, and these species were more effective than the others at 22 and 30 C. Aerobacter cloacae and Paracolobactrum aerogenoides were more effective at 15 C. In general, results were similar with different strains of a species. Except for Aerobacter aerogenes, Klebsiella sp., and P. aerogenoides, which apparently did not compete for nutrients, inhibition of the staphylococcus was by a combination of antibiotic substances and competition for nutrients.  相似文献   

8.
Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10−5 to 2 × 10−6) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.  相似文献   

9.
1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent Ki values being 1·37×10−3−1·67×10−3m for fructose 1-phosphate and 7·2×10−3−7·9×10−3m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent Km values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10−4−1·29×10−4m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent Km values for glucose 6-phosphate were in the range 5·6×10−4−8·5×10−4m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.  相似文献   

10.
Interactions between Bacillus thuringiensis subsp. israelensis and fathead minnows, Pimephales promelas, were studied in laboratory exposures to two commercial formulations, Vectobac-G and Mosquito Attack. Mortality among fatheads exposed to 2.0 × 106 to 6.5 × 106 CFU/ml with both formulations was attributed to severe dissolved oxygen depletion due to formulation ingredients rather than to direct toxicity from the parasporal crystal. No adverse effects were observed at 6.4 × 105 CFU/ml and below. Fathead minnows rapidly accumulated high numbers of spores with 1 h of exposure to 2.2 × 105 CFU of Mosquito Attack per ml, producing whole-body counts of 4.0 × 106 CFU per fish. Comparison of counts on gastrointestinal tract samples and whole-body samples and high numbers of spores in feces indicated that ingestion was the major route of exposure. B. thuringiensis subsp. israelensis spore counts decreased rapidly after transfer of fish to clean water, with a drop of over 3 orders of magnitude in 1 day. Spores were rarely detected in fish after 8 days but were detectable in feces for over 2 weeks. These findings suggest that fish could influence the dissemination of B. thuringiensis subsp. israelensis, and possibly other microbial agents, in the aquatic environment.  相似文献   

11.
Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10−8 to 2 × 10−6, 1 × 10−8 to 4 × 10−8, and <4 × 10−9 to 4 × 10−8 per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10−4 to 1 × 10−3, 9 × 10−4 to 3 × 10−3, and 5 × 10−4 to 4 × 10−3 for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.  相似文献   

12.
Ultrasound-mediated DNA transfer for bacteria   总被引:2,自引:0,他引:2  
In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl2, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 109 CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10−6 transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10−6 and 4.33 ± 0.78 × 10−6 transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.  相似文献   

13.
Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10−19P ≤ 4.5 × 10−18) and D6D (FADS2) (6.05 × 10−8P ≤ 4.20 × 10−7) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10−16). The significance of haplotype association with D5D activity (P = 2.19 × 10−17) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10−28) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10−9) and decreased D6D activity (P = 9.16 × 10−6) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.  相似文献   

14.
A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl2, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 103/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.

Cortical cell protoplasts exhibited energy-dependent uptake of K+ (86Rb), H232PO4, and 36Cl as well as net H+ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K+ was highest between pH 7.5 and 8.0, whereas the influx of H2PO4 was greatest between pH 4.0 and 5.0. K+ and H2PO4 influx and net H+ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl was low, but not greatly different from that observed for other plant cells. K+ flux was somewhat high, probably because the K+ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.

  相似文献   

15.
Total and cellulolytic bacterial and fungal numbers were determined in ruminal and cecal contents of 20 blue duikers (Cephalophus monticola). The animals were equally divided by sex and fed two diets, either high roughage or high concentrate. The mean concentration for total bacterial numbers in the rumen was 26.0 × 108/g of contents, with values ranging from 2 × 108/g to 93 × 108/g. Cellulolytic numbers averaged 6.0 × 108/g with a range of 1.5 × 108/g to 24.0 × 108/g. No differences related to sex or diet were found. In contrast, total bacterial numbers in the cecum differed between diets (P < 0.02), i.e., 1,046 × 106 bacteria per g for animals fed the high-forage diet compared with 166 × 106/g for those fed the high-concentrate diet. Cellulolytic bacterial counts in the cecal contents averaged 3.1 and 7.0% of the total counts for the high-forage and high-concentrate diets, respectively. Low concentrations of fungi were found in both ruminal and cecal contents of some, but not all, animals. Unexpectedly, concentrations of bacteria and fungi in the rumen and cecum were highly correlated with their total numbers (concentration multiplied by total weight of contents).  相似文献   

16.
Curvature, bending moment, and second moment of stem cross-sectional area were evaluated from photographic data and used to compute flexural rigidity and Young's modulus in the panicle rachis of rice, Oryza sativa L. `M-101.' Flexural rigidity C, and its components E, Young's modulus, and I, the moment of inertia of the area about the neutral axis, were evaluated 1.5 cm (tip), 9.5 cm (mid), and 16.5 cm (base) from the tip of the panicle rachis. In dynes per square centimeter, C increases from 1.1 × 103 near the tip to 1.09 × 104 in the middle to 5.35 × 104 in the basal region of the rachis. Of the components of C, the I changes have the larger effect, increasing from 2.12 × 10−7 centimeters4 near the tip to 8.21 × 10−7 centimeters4 in mid regions to 6.0 × 10−6 centimeters4 in the basal regions. Young's modulus increases from 4.8 × 109 dynes per square centimeter near the tip to 1.4 × 1010 dynes per square centimeter in mid regions then falls to 7.4 × 109 dynes per square centimeter near the base of the main stem. Values of Young's modulus from Instron experiments were in satisfactory agreement with values calculated from the beam bending equation. Flexural rigidity in the curved region of the panicle proved independent of panicle load, indicating that the dissected panicle rachis behaves in some respects as a tapered loaded beam.  相似文献   

17.
Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 103 transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 × 10-6 to 7 × 10-6 per donor or recipient.  相似文献   

18.
A new bioassay for auxins and cytokinins   总被引:2,自引:1,他引:2       下载免费PDF全文
The authors have developed a sensitive bioassay that can be used to detect auxins as well as cytokinins. The bioassay is based on the expression in transformed tobacco (Nicotiana tabacum) mesophyll protoplasts of a chimeric gene, consisting of the upstream sequences of the Agrobacterium tumefaciens gene 5, coupled to the coding sequence of the β-glucuronidase. The expression of this gene is induced by the presence of both auxin and cytokinin in the culture medium. Using this assay, indole-3-acetic acid was detected at 5 × 10−8 molar, whereas trans-zeatin could be detected at 5 × 10−11 molar. The assay can be performed in microtiter plates, allowing numerous samples to be analyzed simultaneously. Only 2.5 × 105 protoplasts are required for one individual assay in 250 microliters of culture medium and for qualitative results, the reaction is readily visualized by ultraviolet light.  相似文献   

19.
We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 109 callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10−9 (95% confidence interval = 1.0 × 10−9 − 6.1 × 10−9) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 106. At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10−9 (95% confidence interval = 0.7 × 10−9 − 11 × 10−9).  相似文献   

20.
The rate of cellulose breakdown and density of bacterial populations were measured in the epilimnetic sediments and water columns of lakes in central Ontario that differ in pH, alkalinity, and nutrient status and are particularly sensitive to acidic inputs from atmospheric decomposition. There was no significant difference in decomposition rate in either oxic or anoxic sediment when mean epilimnetic pHs were in the range 5.5 to 6.9. The importance of these findings for the breakdown of autochthonous detritus in Canadian Shield lakes is discussed. Furthermore, the results of these experiments, in which dyed strips of cellophane (regenerated cellulose) were used as substrate, were compared with results of earlier decomposition studies carried out with coarse litter (leaves, twigs). Acridine orange direct counts of bacteria in the top 1 cm of sediment ranged from 5.5 × 108 to 1.0 × 109 per g and in planktonic water samples from 1.1 × 106 to 1.8 × 106 per ml. Bacterial densities were significantly higher in both the shallow sediment (P < 0.01) and the water column (P < 0.05) of dystrophic lakes than at these sites in oligotrophic lakes.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号