Expression in Yeast of a Fusion Gene Composed of the Promoter of a Heat-Shock Gene from Arabidopsis and a Bacterial Gene for {beta}-Glucuronidase

Production of a functional ß-glucuronidase (GUS) proteinwas induced by exposure of exponentially growing yeast cellsto heat shock after transformation of the GUS gene under thecontrol of the promoter of the heat-shock gene, HSP18.2, fromArabidopsis. Yeast cyr and bcy mutations appeared to have essentiallyno effect. ¹Present Address: Laboratory of Plant Molecular Biology, TheRockefeller University, 1230 York Avenue, New York, NY 10021-6399,U.S.A.     

Variation in the DNA Content of Glycine Species

Nuclei were isolated from cotyledons of a range of accessionsfrom 14 species of Glycine. These were stained with ethidiumbromide and the relative fluorescence for each genotype wasmeasured by flow cytometry. The DNA content was estimated bycomparison of relative fluorescence with that from nuclei fromseedling leaves of Allium cepa, whose DNA content has been calculatedpreviously by chemical assay. The 4C amounts for diploid Glycineranged from 3.80 to 6.59 pg. Two groups of diploid species appearedfrom the analysis. The first consisted of species with amountsranging from 3.80 to 5.16 pg and included G. canescens (AA),G. argyrea (A₁ A₁), G. clandestina (A²A²), G. microphylla(BB),G. latifolia (B₁B₁), G. tabacina 2n=40 (B²B²), G. tomentella2n=38 (EE) and 2n=40 (DD), G. max and G. soja (GG), G. arenariaand G. latrobeana. A second group had higher DNA contents rangingfrom 5.27 to 6.59 pg, and consisted of G. curvata, G. cyrtoloba(CC), and G. falcata (FF). The polyploid species, G. tabacina2n=80 (AABB, BBB¹B¹), G. tomentella 2n=78 and 2n=80 (AAEE andDDEE, respectively) contained amounts approximating to the sumsof the respective parental diploid species thought to have givenrise to these allotetraploids. Intraspecific variation was detectedin the DNA content of G. canescens. Within the overall distributionof DNA amounts found in A genome species, each genome containeda range of DNA contents specific to that species. This phenomenonwas also detected amongst B genome species.     

Temporal and spatial distribution of the ichthyotoxic dinoflagellate Gyrodinium aureolum Hulburt in the St Lawrence, Canada

The occurrence of the potentially ichthyotoxic dinoflagellateGyrodinium aureolum Hulburt is reported for the first time inthe Lower Estuary and Gulf of St Lawrence. Taxonomic identificationusing an iramunochemical tagging method suggests a close taxonomicproximity between the species found in the St Lawrence and G.aureolumpresent in European waters. In 1993, G.aureolum was observedalong the entire coast of Quebec and in offshore waters of theGulf of St Lawrence. At the coastal stations, G.aureolum showedlarge week-to-week variations, but higher densities were observedduring late August and early September. Bloom concentrations(10⁶ cells 1^–1) were only observed in the GaspéCurrent at Mont-Louis, on the north coast of the Gaspd peninsula.In the central part of the Gulf, G.aureolum was concentratedin the upper 5 m of the stations directly influenced by thefreshwater run-off of the St Lawrence Estuary. Results fromprincipal component analysis indicate that G.aureolum favorsenvironments with high nitrogen recycling activity.     

Growth Stage Modulates Salinity Tolerance of New Zealand Spinach (Tetragonia tetragonioides, Pall.) and Red Orach (Atriplex hortensis L.)

The response of two speciality vegetable crops, New Zealandspinach (Tetragonia tetragonioides Pall.) and red orach (Atriplexhortensis L.), to salt application at three growth stages wasinvestigated. Plants were grown with a base nutrient solutionin outdoor sand cultures and salinized at 13 (early), 26 (mid),and 42 (late) d after planting (DAP). For the treatment saltconcentrations, we used a salinity composition that would occurin a typical soil in the San Joaquin Valley of California usingdrainage waters for irrigation. Salinity treatments measuringelectrical conductivities (EC_i) of 3, 7, 11, 15, 19 and 23 dSm^-1were achieved by adding MgSO₄, Na₂SO₄, NaCl and CaCl₂to thebase nutrient solution. These salts were added to the base nutrientsolution incrementally over a 5-d period to avoid osmotic shockto the seedlings. The base nutrient solution without added saltsserved as the non-saline control (3 dS m^-1). Solution pH wasuncontrolled and ranged from 7.7 to 8.0. Both species were saltsensitive at the early seedling stage and became more salt tolerantas time to salinization increased. For New Zealand spinach,the salinity levels that gave maximal yields (C_max) were 0,0 and 3.1 dS m^-1and those resulting in a 50% reduction of biomassproduction (C₅₀) were 9.1, 11.1 and 17.4 dS m^-1for early, midand late salinization dates, respectively. Maximal yield ofred orach increased from 4.2 to 10.9 to 13.7 dS m^-1as the timeof salinization increased from 13, to 26, to 42 DAP, respectively.The C₅₀value for red orach was unaffected by time of salt imposition(25 dS m^-1). Both species exhibited high Na⁺accumulation evenat low salinity levels. Examination of K-Na selectivity dataindicated that K⁺selectivity increased in both species withincreasing salinity. However, increased K-Na selectivity didnot explain the increased salt tolerance observed by later salinization.Higher Na-Ca selectivity was determined at 3 dS m^-1in New Zealandspinach plants treated with early- and mid-salinization plantsrelative to those exposed to late salinization. This correspondedwith lower C_maxand C₅₀values for those plants. Lower Ca uptakeselectivity or lower Ca levels may have inhibited growth inyoung seedlings. This conclusion is supported by similar resultswith red orach. High Na-Ca selectivity found only in the early-salinizationplants of red orach corresponded to the lower C_maxvalues measuredfor those plants. Copyright 2000 Annals of Botany Company New Zealand spinach, Tetragonia tetragonioides Pall., red orach, Atriplex hortensis L., salinity, stage of growth, ion accumulation, selectivity, plant nutrition     

Phytoplankton population dynamics of a small reservoir: effect of intermittent mixing on phytoplankton succession and the growth of blue-green algae

The seasonal succession of the phytoplankton of a small reservoir(Guelph Lake, Ontario) in the spring-summer of 1982 was comparedto that in 1981. Mixing of the deeper waters occurred severaltimes throughout the summer in 1982 but not in 1981. The waterat 10 m became anoxic for only 2 weeks in late July in 1982.In contrast, in 1981, the water at 10 m became anoxic at thebeginning of July and remained so until mid-September. The phytoplanktondynamics observed in 1982 did not follow the typical progressionfrom spring diatoms to summer species adapted to survive understratified conditions, as in 1981. Diatoms were present throughoutthe summer in higher amounts in 1982 than in 1981. The mostobvious difference in the two summers was the much greater abundanceof Aphanizomenon flow-aquae in 1982. Other blue-green algaeincluding Microcystis aeruginosa, Gomphosphasria lacustris,and Lyngbya birgei appeared earlier on in 1982, but did notimmediately increase in abundance as in 1981. In 1982, ratesof phytoplankton community change were low in May and June andincreases were observed in mid-July, early August, late Augustand late September. In contrast, in 1981, the rate of communitychange increased in late May, mid-June, early July and lateJuly and remained low throughout August and September. ¹Present address: Department of Zoology, University of Alberta,Edmonton, Alberta T6G 2E1, Canada. ²Present address: CSIRO Division of Fisheries Research, G.P.O.Box 1538, Hobart, Tasmania 7001, Australia     

Effect of nitrate on nitrogen-fixation by the blue-green alga Anabaena cylindrica

The effect of nitrate on nitrogen-fixation in the blue-greenalga Anabaena cylindrica Lemm (Fogg strain) was investigated.At concentrations up to 2x10^–2 M, nitrate neither inhibitedthe activity of nitrogenase nor repressed its formation. Atthe late logarithmic phase, more than 50% of cell nitrogen wasprovided by nitrogen-fixation when the cells were grown in thepresence of nitrate. Ammonia at a concentration of 1x10^–3M completely repressed the formation of nitrogenase, but hadno effect on its activity. Nitrogen-fixing activity in the algavaried to a considerable extent during growth on N₂ and themaximum activity was attained at the middle logarithmic phase.However, atmospheric nitrogen did not directly affect the inductionof nitrogenase. The formation of nitrogenase in A. cylindricaappears to be controlled by the intracellular level of a certainnitrogenous metabolite. ¹ This work was supported by grant No. 38814 from the Ministryof Education. (Received January 26, 1972; )     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

Changes in Nuclear DNA Content, Cell and Nuclear Size, and Frequency of Cell Division in the Cotyledons of Brassica napus L. during Embryogenesis

Cellular behaviour was examined during embryogenesis in Brassicanapus to test whether or not polyploidy occurs in the cotyledonsduring the phase of oil deposition. Nuclear DNA content, nuclearand cell size, and the mitotic index were measured in the cotyledonson various days post anthesis (dpa). In squashed monolayersfrom 15 dpa cotyledons, a polyploid (>5C) population wasdetected together with a substantial number of cells in G2 (4C).Nuclear volume was measured on sectioned tissues and, at 15dpa, the range of values from the cotyledons (40–500 *m³)contrasted with that in the vestigial suspensor and endosperm(50–> 600 µm³). At 15 dpa the nuclear volumedata suggest that whilst cells in the cotyledons were in Gland G2 many endosperm and suspensor cells were polyploid. Thus,polyploidy observed in the squashed monolayers was probablydue to contaminating endosperm/suspensor cells. At 25 and 35dpa, polyploidy was not detected; all cells were in Gl (2C)and cell area increased. The mitotic index peaked at 20 dpabefore declining and given the narrower distribution of nuclearvolumes at 25 and 35 dpa (50–300 µm³), these dataare consistent with cell arrest in Gl. Thus, polyploidy wasnot detected in the cotyledons of B. napus which differs fromwhat is known about cellular development in legume cotyledons. Key words: Brassica napus L., DNA, nuclear volume     

Spatial and temporal aspects of Gyrodinium galatheanum in Chesapeake Bay: distribution and mixotrophy

Gyrodinium galatheanum (Braarud) Taylor 1995 is a common bloom-forming,potentially toxic photosynthetic dinoflagellate in ChesapeakeBay, USA. Abundance of this dinoflagellate achieved densities>4 x 10³ cells ml^–1 in the mid- and upper Bay duringlate spring and early summer of 1995 and 1996. Ingestion ofcryptophytes by this dinoflagellate was detected in most samplescollected from the Bay. During late spring and early summer,mean number of ingested cryptophytes per G.galatheanum was ashigh as 0.46 for dinoflagellate populations located in surfacewaters of the mid- and upper Bay where dissolved inorganic phosphoruswas low. Observations on the distribution of G.galatheanum inChesapeake Bay show that populations of this dinoflagellatewere usually restricted to waters with salinities ranging from7 to 18 psu, seasonally progressed up the estuary, and usuallyco-occurred with cryptophytes. Correlation analysis indicatesthat abundance of G.galatheanum and incidence of feeding wasnegatively correlated with dissolved inorganic phosphorus, andthat incidence of feeding was positively correlated with abundanceof cryptophyte prey. These results indicate that G.galatheanumis an important component of the Chesapeake Bay phytoplanktonduring the spring and summer. Our results suggest that the phagotrophiccapability possessed by this phototrophic dinoflagellate maycontribute to its success in a varying-resource environmentlike Chesapeake Bay.     

Presence of Gymnodinium catenatum (Dinophyceae) in a coastal Mediterranean lagoon

The occurrence and abundance of the toxic, chain-forming dinoflagellateGymnodinium catenatum in a Tyrrhenian coastal lagoon, the Fusaro,during an annual sampling cycle are reported. Peak abundanceswere observed from late spring until early autumn Although veryhigh cell numbers were recorded, up to 1 5 x 10⁶ cells l^–1,no monospecific bloom of this species occurred. The first observationof G.catenatum in the Mediterranean occurred in the Fusaro andthe appearance of this species in a traditional shellfish farmingarea, where no shellfish intoxication has been reported to date,is discussed in relation to human interventions in the basin.In particular, intensive dredging in recent years with resuspensionof bottom sediments may have seeded the water body with cysts.A Gymnodinium n d species, illustrated using scanning electronmicroscopy, caused a monospecific bloom in concomitance withmaximum abundances of G.catenatum, apparently outcompeting thislatter species     

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent ofprotein kinase A. We activated G proteins and monitored CFTRG_Cl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRG_Cl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR G_Cl requiredMg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRG_Cl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa²⁺, suggesting that Ca²⁺-dependent proteinkinase C and Ca²⁺/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with10⁶ M forskolin plus 10⁶ M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathwayin this native absorptive epithelium.

     

Removal by chemicals of photoperiodic light requirements of Lemna gibba G3

Both the initial and the terminal 1 hr portions of the subjectiveday fraction, namely the L1- arid L2-phases, of a 24 hr daymust be illuminated in order for the day to be perceived asa long day in the min-LD determination by the long-day plant,Lemna gibba G3 (9). The light requirement of the L1-phase wassatisfied by a 10 min red light pulse given at the beginningof the phase. The red light effect was erased by a subsequent10 min far-red light, indicating phytochrome-mediated processesoccurring in the L1-phase. The light requirement of the L2-phasewas satisfied by blue or far-red light given during the terminal10 min period of the phase; there was no indication of phytochromeinvolvement. The light action on the L1-phase was replaced by10^–5 M of cyclic AMP or 10^–7 M of DL-isoproterenol.The isoproterenol action was antagonized by 10^–7 M ofDL-propranolol. Cyclic AMP (10^–5 M) combined with salicylicacid (10^–6 M), which can remove the light requirementof the L2-phase (10), rendered a completely dark day physiologicallyequivalent to a long day. Acetylcholine (10^–5 M) exertednyctomimetic action on the L1-phase of the second light day.The action of acetylcholine was antagonized by cyclic AMP (10^–5M). The L2-phase required no light in the presence of 10^–7M of DL-propranolol, and this propranolol action was not affectedby isoproterenol. These findings suggest changes in membranepermeability caused by the light given during the L1- and L2-phases. (Received July 7, 1976; )     

Impermeability of the GIRK2 weaver channel to divalent cations

Asingle amino acid mutation (G156S) in the putative pore-forming regionof the G protein-sensitive, inwardly rectifying K⁺ channelsubunit, GIRK2, renders the conductance constitutively active andnonselective for monovalent cations. The mutant channel subunit(GIRK2wv) causes the pleiotropic weaver disease inmice, which is characterized by the selective vulnerability ofcerebellar granule cells and Purkinje cells, as well as dopaminergicneurons in the mesencephalon, to cell death. It has beenproposed that divalent cation permeability through constitutivelyactive GIRK2wv channels contributes to a rise in internalcalcium in the GIRK2wv-expressing neurons, eventually leadingto cell death. We carried out comparative studies of recombinantGIRK2wv channels expressed in Xenopus oocytes and COS-7cells to determine the magnitude and relative permeability of theGIRK2wv conductance to Ca²⁺. Data from thesestudies demonstrate that the properties of the expressed current differin the two systems and that when recombinant GIRK2wv isexpressed in mammalian cells it is impermeable to Ca²⁺.

     

The Correlation between Cd2+ Sensitivity and Cd2+ Uptake in the Strains of Saccharomyces cerevisiae

The sensitivity of twelve strains of Saccharomyces cerevisiaeto Cd²⁺ was examined in correlation with the uptake of Cd²⁺.Strains of S. cerevisiae were grouped into three categoriesdepending on the sensitivity of cells grown on agar-plates containingvarious concentrations of Cd²⁺. 1) The sensitive group did notgrow in 0.1 mM Cd²⁺. 2) The sub-tolerant group was capable ofgrowth at 0.3 min Cd²⁺, but not at 0.4 mM Cd²⁺. 3) The tolerantgroup was capable of growth at 0.4 mM Cd²⁺ or higher. In thesestrain groups the increase in sensitivity to Cd²⁺ was associatedwith an increase in the activity of Cd²⁺ absorption. ¹ This study is dedicated to the late president J. Ashida ofEhime University. (Received November 25, 1982; Accepted February 14, 1983)     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Model of beta -cell mitochondrial calcium handling and electrical activity. I.Cytoplasmic variables

We continue our development of a kineticmodel of bursting electrical activity in the pancreatic -cell( J. Keizer and G. Magnus. Biophys. J. 56: 229-242,1989), including the influence of Ca²⁺ handling by themitochondria. Our minimal model of mitochondrial Ca²⁺handling [G. Magnus and J. Keizer. Am. J. Physiol. 273 (Cell Physiol. 42): C717-C733, 1997] is expanded toinclude the D-glucose dependence of the rate of productionof mitochondrial reducing equivalents. The Ca²⁺ dependenceof the mitochondrial dehydrogenases, which is also included in themodel, plays only a small role in the simulations, since thedehydrogenases appear to be maximally activated when D-glucose concentrations are sufficient to producebursting. A previous model of ionic currents in the plasma membrane isupdated using a recent experimental characterization of the dependence of the conductance of the ATP-sensitive K⁺(K_ATP) current on adenine nucleotides. The resultingwhole cell model is complex, involving 12 dynamic variables that coupleCa²⁺ handling in the cytoplasm and the mitochondria withelectrical activity in the plasma and inner mitochondrial membranes.Simulations with the whole cell model give rise to bursting electricalactivity similar to that seen in pancreatic islets and clusters ofpancreatic -cells. The full D-glucose dose response ofelectrical activity is obtained if the cytosolic rate of ATP hydrolysisis a sigmoidal function of glucose. The simulations give the correctshape, period, and phase of the associated oscillations in cytosolicCa²⁺, predict that the conductance of the K_ATPcurrent oscillates out of phase with electrical activity [as recentlyobserved in ob/ob mice (O. Larsson, H. Kindmark, R. Bränstrom, B. Fredholm, and P.-O. Berggren. Proc. Natl. Acad.Sci. USA 93: 5161-5165, 1996)], and make other novelpredictions. In this model, bursting results because Ca²⁺uptake into mitochondria during the active phase reduces the mitochondrial inner membrane potential, reducing the rate of production of ATP, which in turn activates the K_ATP current andrepolarizes the plasma membrane.

     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)