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Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants 总被引:7,自引:1,他引:6
The expression of foreign genes in pollen may pose potentialproblems in the field release of transgenic plants, since pollenrepresents a route whereby foreign genes and their productsmay escape into the wider environment. The possible risks posedby cross-hybridization with wild relatives have been extensivelyexplored, but problems that may arise due to the expressionof foreign gene products in pollen have not been so widely studied.The activities of the CaMV 35S and nos promoters in pollen inpopulations of stably transformed plants and in transient expressionanalysis are described. These promoters are commonly used inall areas of plant molecular biology research and their expressionpatterns will be of interest to those involved in field releasestudies. The results show that both promoters had no detectablepollen activity in Arabidopsis, but both showed activity intobacco pollen. The CaMV 35S-gus gene fusion showed heritableexpression levels in tobacco pollen of up to a maximum of 64.6pmol 4-MU min1 mg 1 total protein. nos promoteractivity in transgenic tobacco pollen was highly variable, withGUS activities ranging from undetectable levels up to 2561 pmol4-MU min1 mg1 total protein within the transgenicpopulation. Histochemical staining of anther sections from 1012mm buds revealed that the CaMV 35S promoter had some activityin the vascular bundle, stomium and tapetum, while GUS expressionfrom the nos promoter in sporophytic tissues was confined entirelyto the stomium. Key words: CaMV 35S promoter, nos promoter, pollen, transgenic plant release 相似文献
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Chlorophyll (Chl) a was measured every 10 m from 0 to 150 min the Transition Domain (TD), located between 37 and 45°N,and from 160°E to 160°W, in May and June (Leg 1) andin June and July (Leg 2), 199396. Total Chl a standingstocks integrated from 0 to 150 m were mostly within the rangeof 20 and 50 mg m2. High standing stocks (>50 mg m2)were generally observed westof 180°, with the exceptionof the sporadic high values at the easternmost station. Thetotal Chl a standing stock tended to be higher in the westernTD (160°E172°30'E) than in the central (175°E175°W)and eastern (170°W160°W) TD on Leg 1, but thesame result was not observed on Leg 2. It was likely that largephytoplankton (210 and >10 µm fractions) contributedto the high total Chl a standing stock. We suggest that thehigh total Chl a standing stock on Leg 1, in late spring andearly summer, reflects the contribution of the spring bloomin the subarctic region of the northwestern Pacific Ocean. Thedistribution of total Chl a standing stock on Leg 2 was scarcelyaffected by the spring phytoplankton bloom, suggesting thattotal Chl a standing stock is basically nearly uniform in theTD in spring and summer. Moreover, year-to-year variation inthe total Chl a standing stock was observed in the western TDon Leg 1, suggesting that phytoplankton productivity and/orthe timing of the main period of the bloom exhibits interannualvariations. 相似文献
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The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) 226 and 54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt 226 to 151and from nt 84 to 54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt 226 to 151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt 226 to 84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996) 相似文献
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The effects of the chemical modification of tryptophan residuesin native pea (Pisum sativum L.) phytochrome by 2-hydroxy-5-nitrobenzylbromide (HNB-Br) were examined. Such treatment had no effecton the spectral properties or on the pattern of tryptic digestionof phytochrome, which indicated that no major conformationalchange in phytochrome had occurred. Amino acid analysis of theHNB-Br-treated phytochrome indicated that the number of modifiedTrp residues after the treatment was dependent on the light-absorbingform. The values were three for PR and five for PFR (out ofa total of ten) per monomer. The results indicate that two additionalTrp residues are exposed on the molecular surface of PFR whenthe photoconversion of PR to PFR occurs. The amino acid analysisof a 58-kDa tryptic fragment of phytochrome (a mixture of peptides,residues 63583 and 66587) showed that one Trpresidue in the fragment from PR and two in that from PFR (outof six) were modified by HNB-Br. In the 56-kDa fragment (a mixtureof peptides, residues 5981121 and 6031124), therewere two modified Trp residues in PR and three in PFR (out offour). The Trp residue in a 36-kDa fragment (residues 66383),which includes the tetrapyrrolic chromophore, was not modifiedin the either case. These results indicate that new exposedsites that are generated by the photoconversion of PR to PFRare in the region between Trp456 and Trp567 andin that between Trp644 and Trp787. (Received February 25, 1993; Accepted August 16, 1993) 相似文献
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Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1 总被引:2,自引:0,他引:2
Akel A Wagner CA Kovacikova J Kasinathan RS Kiedaisch V Koka S Alper SL Bernhardt I Wieder T Huber SM Lang F 《American journal of physiology. Cell physiology》2007,292(5):C1759-C1767
Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl removal, and energy depletion activate Ca2+-permeable cation channels with Ca2+-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1/ mice) than in their wild-type littermates (AE1+/+ mice) despite increased percentages of reticulocytes (AE1/: 49%, AE1+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1/erythrocytes/reticulocytes (10%) than in AE1+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1/ erythrocytes/reticulocytes than in AE1+/+ erythrocytes. The increase of annexin binding following exposure to the Ca2+ ionophore ionomycin (1 µM) was, however, similar in AE1/ and in AE1+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca2+ permeability in AE1/ erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1/ mice than in AE1+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca2+ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion 相似文献
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Suzuki Masaharu; Koide Yasuhiro; Hattori Tsukaho; Nakamura Kenzo; Asahi Tadashi 《Plant & cell physiology》1995,36(6):1067-1074
Deletion analysis of the promoter region of a gene for catalase,cat2, from castor bean (Ricinus communis) was performed to identifythe cis-regulatory elements responsible for the expression ofa rß-glucuronidase (GUS) fusion gene during seed formationand postembryonic development in transgenic tobacco. The analysisshowed that multiple cis-elements contribute to the activityof the cat2 promoter during seed formation and postembryonicdevelopment. The 5'-upstream regions from 1,241 to 816bp, from 720 to 682 bp, and from 632 to535 bp, relative to the site of initiation of translationof cat2, contributed positively to the activity of the cat2promoter during both stages. By contrast, the region from 816to 720 bp had a negative effect at both stages. The regionfrom 682 to 632 bp contributed positively to theactivity during seed formation but negatively during postembyonicdevelopment. Histochemical analysis revealed that the multiplecis-elements determined not only the level of expression ofthe chimeric gene but also the tissue-specificity of such expression.For example, the region from 1,241 to 816 bp allowedexpression of the chimeric gene in the axis of the embryo ofthe dry seed, as well as in the cortex of the middle part ofthe hypocotyl and at the base of epicotyl in the young seedling.
1Present address: Department of Plant Molecular and Cell Biology,University of Florida, Gainesville, Florida 32611-0511, U.S.A.
2Present address: Center for Molecular Biology and Genetics,Mie University, 1515 Kamihama, Tsu, Mie, 519 Japan
3Present address: Faculty of Biotechnology, Fukui PrefecturalUniversity, 4-1-1 Kenjojima, Matsuoka-cho, Yoshida-gun, Fukui,910-11 Japan 相似文献
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During nitric oxide signaling, type I cGMP-dependent protein kinase (PKGI) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGI interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH2-terminal LZ of PKGI (HK Surks and ME Mendelsohn. Cell Signal 15: 937944, 2003; HK Surks et al. Science 286: 15831587, 1999), but we previously showed that PKGI interacts with LZ-positive (LZ+) and LZ-negative (LZ) MYPT1 isoforms (13). Interestingly, PKGI is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 1477214777, 2000), and there is an RK motif within the aa 888928 sequence of MYPT1 in LZ+ and LZ isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI interaction, we designed four MYPT1 fragments that contained both the aa 888928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916917 (R916K917) to AA decreased binding of MYPT1 to PKGI in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R916K917 to E916E917 eliminated binding, suggesting that one factor important for the PKGI-MYPT1 interaction is the charge at aa 916917. These results suggest that, during cGMP-mediated signaling, aa 888928 of MYPT1 mediate the PKGI-MYPT1 interaction. myosin light chain phosphatase; nitric oxide; smooth muscle; calcium desensitization; cGMP-dependent protein kinase; cGMP 相似文献
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Planktonic primary production in the German Wadden Sea 总被引:8,自引:0,他引:8
By combining weekly data of irradiance, attenuation and chlorophylla concentrations with photosynthesis (P) versus light intensity(E) curve characteristics, the annual cycle of planktonic primaryproduction in the estuarine part of the Northfrisian WaddenSea was computed for a 2 year period. Daily water column particulategross production ranged from 5 to 2200 mg C m2 day1and showed a seasonal pattern similar to chlorophyll a. Budgetcalculation yielded annual gross particulate primary productionsof 124 and 176 g C m2 year1 in 1995 and 1996,respectively. Annual amounts of phytoplankton respiration, calculatedaccording to a two-compartment model of Langdon [in Li,W.K.W.and Maestrini,S.Y. (eds), Measurement of Primary Productionfrom the Molecular to the Global Scale. International Councilfor the Exploration of the Sea, Copenhagen, 1993, pp. 2036],and dissolved production in 1996, were both in the range of2439 g C m2 year1. Annual total net productionwas thus very similar to particulate gross production (127 and177 g C m2 year1 in 1995 and 1996, respectively).Phytoplankton growth was low or even negative in winter. Inspring and summer, production/biomass (Pr/B) ratios varied from0.2 up to 1.7. Phytoplankton growth during the growth seasonalways surpassed average flushing time in the area, thus underliningthe potential of local phytoplankton bloom development in thispart of the Wadden Sea. The chlorophyll-specific maximum photosyntheticrate (PBmax) ranged from 0.8 to 9.9 mg C mg1 Chl h1and was strongly correlated with water temperature (r2 = 0.67).By contrast, there was no clear seasonal cycle in B, which rangedfrom 0.007 to 0.039 mg C mg1 Chl h1 (µmolphotons m2 s1)1. Its variability was muchless than PBmax and independent of temperature. The magnitudeand part of the variability of PBmax and B are presumably causedby changes in species composition, as evidenced from the rangeof these parameters found among 10 predominant diatom speciesisolated from the Wadden Sea. The ratio of average light conditionsin the water column (Eav) to the light saturation parameterEk indicates that primary production in the Wadden Sea regionunder study is predominantly controlled by light limitationand that nutrient limitation was likely to occur for a few hoursper day only during 5 (dissolved inorganic nitrogen) to 10 (PO4,Si) weeks in the 2 year period investigated. 相似文献
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Alexander Edward A.; Brown Dennis; Shih Theodora; McKee Mary; Schwartz John H. 《American journal of physiology. Cell physiology》1999,276(3):C758
In previousstudies, our laboratory has utilized a cell line derived from the ratinner medullary collecting duct (IMCD) as a model system for mammalianrenal epithelial cell acid secretion. We have provided evidence, from aphysiological perspective, that acute cellular acidification stimulatesapical exocytosis and elicits a rapid increase in proton secretion thatis mediated by an H+-ATPase. Thepurpose of these experiments was to examine the effect of acutecellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro.We utilized the 31-kDa subunit of theH+-ATPase as a marker of thecomplete enzyme. The distribution of this subunit of theH+-ATPase was evaluated byimmunohistochemical techniques (confocal and electron microscopy), andwe found that there is a redistribution of these pumps from vesicles tothe apical membrane. Immunoblot evaluation of isolated apical membranerevealed a 237 ± 34% (P < 0.05, n = 9) increase in the 31-kDa subunitpresent in the membrane fraction 20 min after the induction of cellularacidification. Thus our results demonstrate the presence of this pumpsubunit in the IMCD cell line in vitro and that cell acidificationregulates the shuttling of cytosolic vesicles containing the 31-kDasubunit into the apical membrane. 相似文献
13.
A Far-Upstream Sequence of the Wheat Histone H3 Promoter Functions Differently in Rice and Tobacco Cultured Cells 总被引:2,自引:0,他引:2
Ito Takuya; Fujimoto Yoshinobu; Nakayama Takuya; Iwabuchi Masaki 《Plant & cell physiology》1995,36(7):1281-1290
The cis-regulatory function of a far-upstream sequence (1,711to 186) of the promoter of the wheat gene for histoneH3 (TH012) was analyzed in cultured rice and tobacco cells ina transient expression system with the gene for rß-D-glucuronidaseas a reporter gene. The far-upstream sequence was necessaryfor full activity of the H3 promoter in rice cells but did notenhance the activity of the proximal promoter in tobacco cells.Dissection analysis of the far-upstream sequence revealed theexistence of several positive and negative cis-acting sequencesin this region, some of which functioned differently in riceand tobacco cells. In gain-of-function experiments with ricecells, the sequence from 848 to 704, containingthe CCAAT and octamer (CaCGGATC) motifs, functioned in an orientation-independentmanner, whereas the sequence from 703 to 486 functionedin an orientation-dependent manner. By contrast, both sequencesexhibited an orientation-dependent cis-function in tobacco cells.These findings suggest that some cis-regulatory sequences inthe far-upstream region of the H3 promoter function differentlyin rice and tobacco cells. (Received May 10, 1995; Accepted August 8, 1995) 相似文献
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Organic carbon release by phytoplankton: its composition and utilization by bacterioplankton 总被引:7,自引:0,他引:7
The present study characterized the rate of production of extracellularlyreleased organic carbon (ROC) by phytoplankton, its molecularweight distribution, subsequent utilization and transformationby bacterioplankton in situ. Primary production rate of phytoplanktonwas high during the study due to continuous blooms of smalldinoflagellates and ranged from 59.8 to 298.7µg CI1h1. The rate of organic carbon release varied from 1.3to 123.7 µg CI1 h1 and constituted from4.0 to 68.9% of the total carbon fixed in photosynthesis. TheROC was fractionated on molecular weight (MW) basis. A low MWfraction less than 500 daltons (18.5% of ROC), a fraction ofMW 10 00030 000 daltons (30% of ROC), and high MW fractionof > 300 000 daltons (15.4% of ROC), were the most dominantin ROC. Bacterioplankton utilized a significant portion of ROC,ranging from 18 to 77%. Part of the utilized ROC incorporatedby the bacterioplankton (31 56%), and the remainderwas respired (mineralized). ROC not utilized by bacteria wascomposed of high MW compounds. The dynamics of the in situ utilizationof ROC and its role as a link between autotrophic and heterotrophicprocesses in the estuary are described.
1Present address: Department of Environmental Microbiology,Institute of Microbiology, Warsaw University, Warsaw, Poland 相似文献
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Results are presented of size-fractionated primary productionstudies conducted in the vicinity of the Subtropical Front (STF),an adjacent warm-core eddy, and in Sub-antarctic waters duringthe third South African Antarctic Marine Ecosystem Study (SAAMESIII) in austral winter (June/July) 1993. Throughout the investigation,total chlorophyll (Chl a) biomass and production were dominatedby small nano- and picophytoplankton. No distinct patterns intotal Chl a were evident. At stations (n = 7) occupied in thevicinity of the STF, total integrated biomass values rangedfrom 31 to 53 mg Chl a m2. In the vicinity of the eddy,integrated biomass at the eddy edge (n = 3) ranged from 24 to54 mg Chl a m2 and from 32 to 43 mg Chl a m2 inthe eddy (n = 2). At the station occupied in the Sub-antarcticwaters, total integrated biomass was 43 mg Chl a m2.Total daily integrated production was highest at stations occupiedin the vicinity of the STF and at the eddy edge. Here, totalintegrated production ranged from 150 to 423 mg C m2day1 and from 244 to 326mg C m2 day1, respectively.In the eddy centre, total integrated production varied between134 and 156 mg C m2 day1. At the station occupiedin the Sub-antarctic waters, the lowest integrated production(141 mg C m2 day1) during the entire survey wasrecorded. Availability of macronutrients did not appear to limittotal production. However, the low silicate concentrations duringthe survey may account for the predominance of small nano- andpicophytoplankton. Differences in production rates between theeddy edge and eddy core were related to water column stability.In contrast, at stations occupied in the vicinity of the STF,the control of phytoplankton production appears to be relatedto several processes, including water column stability and,possibly, iron availability. 相似文献
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The arcA, a member of the G protein rß-subunit family,was isolated from tobacco BY-2 cells as an auxin-responsivegene. Characterization of arcA, which should help to elucidatethe function of the gene product in the plant cells, was performedwith emphasis on the mode of expression and the analysis ofits promoter. Accumulation of the arcA message was detectedonly after treatments with auxins and not after treatments withother phytohormones or CdCl2, implying that responsiveness ofarcA was exclusive to auxin. The putative arcA promoter regionwas fused to a reporter gene for rß-glucuronidase(GUS), and transient expression was analyzed in tobacco BY-2cells. Two series of arcA promoter/GUS chimeric genes were constructed.One consisted of a set of 5' nested deletions of the arcA promoterconnected to the gene for GUS and the other consisted of a varietyof the arcA promoter fragments fused to a minimal promoter-GUSconstruct. The results indicated that the promoter sequencecovering four sets of direct repeats ( 562 to 167)was necessary for the sufficient response of arcA promoter toauxin in BY-2 cells. Moreover, irrespective of auxin treatment,elevated activity of GUS driven by this promoter fragment wasdetected, a result that implies that this region behaves anenhancer in BY-2 cells. (Received September 30, 1995; Accepted March 1, 1996) 相似文献
18.
BERLIN J.; WRAY V.; FORCHE E.; RENG H.-G.; SCH?LER H.; LUCKINGER R.; MOHLBACH H.-P. 《Journal of experimental botany》1985,36(12):1985-1995
Berlin, J., Wray, V., Forche, E., Reng, H.G , Schler,H, Luckinger, R. and Mhlbach, H.P. 1985. Production ofpotato spindle tuber viroid (PSTV) by large scale fermentationof PSTVinfected potato cell suspension cultures.J.exp. Bot 36: 19851995. Cell suspension cultures of Solatiumdemissum, infected with the potato spindle tuber viroid (PSTV),were scaled up to volumes of up to 800 dm3 to provide sufficientand uniform plant material for subsequent studies on viroidbiosynthesis. Here we describe the technological aspects ofproducing the required amounts of biomass and viroid. The cells,which had been maintained on a medium containing expensive coconutmilk, were first adapted to rapid growth on the less expensiveB5medium. The physiological state of the cells was monitoredby in vivo 31PNMR spectroscopy Under the chosen conditionsthe scaleup from 10 dm3 inoculum from shake flasks tothe harvest of the 800 dm3 stirred fermenter lasted 38 d andprovided 112 kg biomass. Growth characteristics and viroid productionin shake flasks and large bioreactors were rather similar. Gelelectrophoretic analysis of isolated nucleic acids using silverstaining and Northern blot hybridization revealed a PSTVcontentof approximately 700 µg PSTV per kg fresh mass of culturedcells. Key words: Solanum demissum, plant cell cultures, potato spindle tuber viroid, biomass production, fermentation, in vivo 31P-NMR 相似文献
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Biermann J Lang D Gorboulev V Koepsell H Sindic A Schröter R Zvirbliene A Pavenstädt H Schlatter E Ciarimboli G 《American journal of physiology. Cell physiology》2006,290(6):C1521-C1531
Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (55 ± 5%, n = 10 and 63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (54 ± 5%, n = 14, and 31 ± 6%, n = 26) and PKA (16 ± 5%, n = 16, and 18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (16099 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride 相似文献