Direct Determination of NotI Cleavage Sites in the Genomic DNA of Adult Mouse Kidney and Human Trophoblast Using Whole-Range Restriction Landmark Genomic Scanning

Restriction landmark genomic scanning (RLGS) is a method forvisualizing restriction landmarks, employing direct labelingof restriction sites of genomic DNA and high-resolution two-dimensionalelectrophoresis. We determined the conditions for both the firstand second dimensions of RLGS that define all of the restrictionfragments which carry the NotI landmark. Using this system,we determined the number of cleavable NotI sites of genomicDNA from the mouse kidney (C57BL/6) and from the human placenta.The mouse and human genomes were cleaved at 2,380±80sites (4,760±160 spots) and 3,240±110 sites (6,480±220spots), respectively with NotI.     

Cloning and Characterization of New Repetitive Sequences in Field Bean (Vicia fabaL.)

Five new repetitive sequences have been isolated from theViciafabagenome, by cloning bands visible on agarose gel electrophoresisafter digestion of genomic DNA with various restriction enzymes.The sequences were 109 to 584 bp long, their abundance rangingfrom 5x10⁴to 5x10⁵copies per haploid genome. Southern blot andinsituhybridization revealed that four of five newly isolatedrepeats were dispersed in theV. fabagenome. One of the repeats(TIII15) showed tandem organization with several major hybridizationspots on mitotic chromosomesin situ.These sites were distributedin euchromatic as well as in heterochromatic chromosomal regions,and in several loci they were simultaneously localized withpreviously describedFokI repeated elements. The sequence ofTIII15 comprises four 26–27 bp subrepeats, but sharesno homology toFokI elements which have similar sequence organization.All newly described sequences were highly specific forV. faba,withlittle or no hybridization to DNA of otherViciaspecies, andno hybridization to DNA of other legumes tested.Copyright 1999Annals of Botany Company Vicia faba, field bean, repeated DNA sequences, FISH, PRINS, genome organization, copy number.     

Bacterivory in seawater samples estimated by a dual radioactive-labelling technique

A dual radioactive-labelled bacteria technique using Vibrio(DRLV), developed for laboratory studies on bacterivory, hasbeen refined for use at the concentrations of prey and predatorstypcially found at sea. Experiments with estuarine water collectedin spring and in autumn showed that bacterivorous nanoflagellates(HNF) (concentration 1.38±0.35x10³ HNF ml^–1) ingested2.7±0.96 DRLV flagellate1^–1 h^–1 at concentrationsof 0.8–2.2x10⁶ DRLV ml^–1 in the presence of 2.04±0.68x10⁶natural bacteria ml^–1. The method was also applied tosamples collected in October in the Celtic Sea, when on average1 ml of water from the surface layer contained 1.41±0.16x10⁶natural bacteria, 14.6x10³ cyanobacteria, 530±170 HNF,7.3±3.0x103 phototrophic nanoflagellates (1.5–4µm), 49.0±26.5 phototrophic dinoflagellates, 36.3±12.6heterotrophic dinoflagellates and 21.3±9.5 Leucocryptosmarina. Under these conditions the grazing rate in most samplesdid not exceed the coefficient of variation of the method (2%),although we estimate the grazing rate was -1.6 DRLV HNF^–1h^–1 and on one occasion a rate of 2.45 was recorded. Thegross growth efficiency for protein of -30% displayed by naturalHNF means that they could release about     

The Genetic Basis of Preference for Sweet Substances Among Inbred Strains of Mice: Preference Ratio Phenotypes and the Alleles of the Sac and dpa Loci

Five inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BI/6J andDBA/2J) were examined with two-bottle (48 h) preference ratiotesting across concentrations of sodium saccharin (3 x 10^–4M, 10^–3 M, 3 x 10^–3 M and 10^–2 M), d-phenylalanine(10^–3 M, 10^–2 M and 10^–1 M), and l-glutamine(10^–2 M, 3 x 10^–2 M, 10^–1 M and 3 x 10^–1M). Three consistent groupings of strains were observed acrosssubstances and concentrations:

1. C57BI/6J (preference at low andhigh concentrations);
2. BALB/cByJ and C3HeB/FeJ (preferenceat high concentrations);
3. 129/J and DBA/2J (preference at highconcentration for sodiumsaccharin and indifference to d-phenylalanineand l-glutamine).

If a single locus (presumably dpa or Sac) determines these phenotypes,there are likely to be three alleles. If two independent loci(presumably dpa and Sac) determine these phenotypes, an allelicassignment of Sac^b/dpa^+s for the C57BI/6J strain, Sac^b/dpa^–sfor the BALB/cByJ and C3HeB/FeJ strains, and either Sac^d/dpa^+sor Sac^d/dpa^–s for the 129/J and DBA/2J strains is suggested.Chem. Senses 20: 291–298, 1995.     

L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)     

Behavioral determinations of thresholds for n-butyl acetate and nbutyl alcohol in the tiger salamander (Ambystoma tigrinum)

Two groups of tiger salamanders (Ambystoma tigrinum) were conditionedto respond to odorant-air mixtures of n-butyl acetate (8.9 x10^–5M) or n-butyl alcohol (6.7 x l0^–5M). They werethen given tests with various concentrations of the trainingodorants presented using a temporal forced-choice method ofascending limits. Results showed that reliable responses toodorant-air presentations were obtained with concentrationsof n-butyl acetate above 2.4 x l0^–7M and with concentrationsof n-butyl alcohol above 8.5 x 10^–8M. These results arein substantial agreement with previous dectrophysiological findings.     

An automatic image analysis system for RLGS films

The RLGS (Restriction Landmark Genome Scanning) method was originally developed as a powerful method for enabling viewing of thousands of restriction landmarks. It offers a tool for obtaining information about genetic loci, with a single RLGS profile displaying approximately 2000 restriction landmarks as spots. One of the most useful applications is RLGS spot mapping, which allows the efficient, low-cost construction of the genetic map of any organism. However, analyses of the profiles depend mainly on human visual observation and are tedious and laborious. Although several commercially available image analyzing systems for profile comparison have been examined, they cannot be used for the RLGS spot mapping system owing to the background characteristics of the RLGS profiles, unsatisfactory rates of correspondence, and inefficient correction of informative genetic data. We therefore developed a novel automatic image analysis system for RLGS spot mapping, using an original algorithm based on the binary image transferred from the original RLGS profile. This system was employed for identifying non-polymorphic and parental strain-specific polymorphic spots of the F₁ mouse profile and yielded efficient initial screening of RLGS profiles. Received: 5 December 1997 / Accepted: 6 April 1998     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Effects of algal concentration, bacterial size and water chemistry on the ingestion of natural bacteria by cladocerans

Journal of Plankton Research, 11, 1273–1295, 1989. The values of P/U⁰ (Table I) and fluid velocity used to calculatethe energy required for sieving (pp. 1289–1290) and severalequations (footnote b of Table I; p. 1290, lines 3–4)are incorrect. The corrected table appears below: Table I. Filter setule measurements (mean and within specimenstandard deviation) of the gnathobases for the cladocerans studied^aGnathobaseof trunklimb number. ^bP = 8µU₀/(b(1 – 21nt + 1/6(t²) - 1/144(t⁴))), whereP = pressure drop in dyn cm^–2, =3.1416, U₀ = fluid velocityin cm s^–1, b = distance between setule centres in cm,t = ( x setule diameter)/b and µ = 0.0101 dyn s^–1cm^–2. Formula from Jørgensen (1983). The text (p. 1289, line 19 to p. 1290, line 10) should read: organism. Using a similar argument, a 0.5 mm Ceriodaphnia witha filter area of 0.025 mm² (Ganf and Shiel, 1985) and pressuredrop P = 2757 dyn cm^–2 (with fluid velocity of 0.07 cms^–1) allocates only 2171 ergs h^–1 to filtrationof a total energy expenditure of 10⁴ ergs h^–1 [filtrationenergy (ergs h^–1) = area (cm²) x pressure drop (dyn cm^–2)x 3600 (s h^–1) x 1/0.2 (efficiency of conversion of biochemicalinto mechanical work); total energy (ergs h^–1) = respiration(0.05 µl O₂ ind^–1 h^–1 consumed; Gophen, 1976)x conversion factor (2 x 10⁵ ergs µl^–1 O₂). Withan estimated 0.034 mm² in filter area, fluid velocity of 0.041cm s^–1 and respiration of 1.8 x 10⁴ ergs h^–1 (calculatedfrom Porter and McDonough, 1984), a 0.5 mm Bosmina uses <4%of its metabolism to overcome filter resistance. The velocities used in the original examples (0.4 cm s^–1for Ceriodaphnia, 0.2 cm s^–1 for Bosmina) were derivedfrom literature values of appendage beat rate and estimatesof the distance travelled by the appendages during each beatcycle. This approach unnecessarily assumes that all water movedpasses through the filter. In the new calculations, the flowacross the filter needed for food to be collected by sieving(0.07 cm s^–1 for Ceriodaphnia and 0.041 cm s^–1 forBosmina) was determined from the maximum clearance rate/filterarea. The amended energy expenditures, although higher, do notrefute the sieve model of particle collection.     

The Effects of Light Intensity and External Potassium Level on Root/Shoot Ratio and Rates of Potassium Uptake in Perennial Ryegrass (Lolium perenne L.)

Loliun perenne L. (cv.S. 23) was grown on vermiculite in winterin a heated greenhouse for 8 weeks under factorial combinationsof two potassium regimes (nominally 6 parts/10⁶ and 156 parts/10⁶in Hewitt's solution) and three densities of artificially supplementedvisible radiation flux (36.1, 7.3, and 2.2 W m^–2). Growthand potassium uptake were studied through the calculation ofvarious growth functions from fitted curves. There was little effect of potassium treatment but the experimentalmaterial responded markedly to light. Leaf-area ratio in thethree treatments showed extreme plasticity in increasing from2–3 x 10^–2 through 6 x 10^–2 to 8–9 x10^–2 m² g^–1 as light intensity decreased. Correspondingdecreases in unit leaf rate, however, caused over-all reductionsin relative growth rate. Specific absorption rates for potassium (A_K, dry-weight basis)were strongly reduced at the lower light intensities but alsodisplayed complex ontogenetic drifts. Values of the allometricconstant, k (the ratio of root and shoot relative growth rates),decreased from c. 0.7 at 36.1 W m^–2 through c. 0.3 at7.3 W m^–2 to a value not significantly different fromzero (P < 0.05) at 2.2 W m^–2. In material grown under the two higher light intensities a constantinverse relationship was found between the mass ratio of rootand shoot and the corresponding activity ratio. The resultsconform to this model: Mass ratio = –0.001+45.0 (1/activityratio) where activity ratio is expressed as specific absorptionrate for potassium (in µg g root^–1 h^–1)/unitshoot rate (rate of increase of whole-plant dry weight per unitshoot dry weight, in mg g shoot^–1 h^–1). The implicationsof this relationship are discussed.     

Diffusion of Oxyleghaemoglobin

The diffusion of oxyleghaemoglobin, prepared from soybean rootnodules, was measured at 24°C in agar and agarose gels ofvarious strengths, or in 1% agarose containing 0-18% (w/v) bovineserum albumin, to simulate the protein content of the cytoplasmof root nodule cells. Values of D_p, the diffusion coefficient,were unaffected (D_p = 11·8 x 10^-11 m² s^-1; s.e.m. 0·3x 10^-11) until the protein concentration exceeded 6%, abovewhich D_p declined sharply. With 18% bovine serum albumin, theconcentration of total soluble protein calculated to be presentin the cytoplasm of infected cells, where most of the leghaemoglobinis located in vivo, D_p was 5·9 x 10^-11 m² s^-1. Theseresults are discussed in relation to leghaemoglobin-facilitateddelivery of O₂ to the respiring N₂-fixing bacteroids in rootnodule cells.Copyright 1993, 1999 Academic Press Bacteroids, diffusion, Glycine max, N₂ fixation, oxyleghaemoglobin, soybean, root nodules     

The Organization of rRNA Genes in Vicia faba

Vicia faba DNA was digested with restriction endonucleases andfractionated on 1% agarose gels. The physical map for EcoRI,BamHI and XbaI cleavage sites in V. faba cytosol rDNA was determinedusing the Southern blot hybridization technique. XbaI and BglIIdigestion or partial EcoRI digestion showed that the lengthof a major repeat unit of V. faba rDNA is 6.7 x 10⁶ daltons.EcoRI and BamHI cleaved this unit into two and five DNA fragments,respectively. (Received April 23, 1981; Accepted July 20, 1981)     

Effect of Spermidine and Methylglyoxal-bis (Guanyl-hydrazone) (MGBG) on In Vitro Pollen Germination and Tube Growth in Catharanthus roseus

Incorporation of polyamine-spermidine into the nutrient mediumat 10^–6 and 10^–5 M concentrations stimulates pollen-tubegrowth in vitro in Catharanthus roseus L. G. Don. MGBG, an inhibitorof spermidine biosynthesis, at 0.5 x 10^–3 and 1 x 10^–3M concentrations reduced the percentage of germination as wellas tube growth and at a concentration of 1.5 x 10^–3 Mgermination was totally inhibited. Pollen grains incubated inthe medium containing 1.5 x 10^–3 M MGBG, when transferredto a fresh medium with 10^–5 M spermidine, resulted in80% germination recovery, along with considerable tube growth.Experiments with actinomycin-D indicate that stimulation ofpollen-tube growth by spermidine may involve de novo synthesisof protein. Catharanthus roseus, pollen germination, tube growth, spermidine, MGBG, inhibition, actinomycin-D     

Effect of temperature and food concentration on post-embryonic development, egg production and adult body size of calanoid copepod Eurytemora affinis

Post-embryonic development time, egg production rate and adultbody size of calanoid copepod Eurytemora affinis from Lake Ohnuma,Japan, were determined under six temperature-food conditions(10³,5 x 10³, 10⁴ and 5 x 10⁴ cells ml^–1 at 15°C,5 x 10⁴ cells ml^–1 at 10and20°C) in the laboratory.The measured parameters varied with both temperature and foodconcentration. Development time from hatching to adult femalewas 9.2, 11.4 and 22.8 days at 20, 15 and 10°C respectively,at the highest food concentration. The males developed to adultat one to two days earlier than the females. An effect of foodshortage on development time occurred at the lowest food concentration.This development time was 24.8 days even at 15°C, beingtwice as long as that at the highest food concentration. Prosomelength of these food-limited females was approximately 75% ofwell-fed ones, which reduced by only 10% with increasing temperaturefrom 10 to 20°C. Clutch size (C, eggs clutch^–1) ofwell-fed individuals depended on prosome length of the adultfemale (L, mm), and was expressed as an equation: C = 65.2 L³⁸³. Clutch size of individuals reared at less than 10⁴ cellsml^–1, however, mostly laid below the estimated curve,especially at the lowest food concentration being only 10% ofthat at the highest food concentration. These results suggestthat food availability is a more important factor affectingpopulation growth of E.affinis in Lake Ohnuma than variationof temperature.     

Regulation of Free Calcium Concentration in the Pitchers of the Carnivorous Plant Sarracenia purpurea: A Model for Calcium in the Higher Plant Apoplast?

The pitcher of the carnivorous plant Sarracenia purpurea L.contains an entrapped body of liquid within which its prey isdigested. Free calcium in the pitcher is derived from eitherthe pitcher walls or from prey falling into the pitcher; inthe absence of exogenous (prey-derived) calcium it will dependon the active and passive calcium regulatory properties of thepitcher walls and may to some extent therefore mimic calciumin the apoplast of plant cells. Using a calcium-specific electrode,the free calcium concentration of the pitchers of Sarraceniaplants was investigated and the effect of adding a variety ofconcentrations of calcium in water determined. The mean pitcherfree calcium concentration in vivo was 2.3 x 10^–5 M±2.5x 10^–5 M; when pitchers were washed and filled with watercontaining lower calcium concentrations, the concentration inthe pitcher water rose to 1–5 x 10^–5 M. When highercalcium concentrations (up to 1 x 10^–4 M) were added,the pitcher calcium concentration declined to 1–7 x 10^–5M. Concentrations of calcium above 1 x 10^–4 M were alsoreduced, but to a lesser extent. Metabolic inhibition of activeion transport, while inhibiting pitcher acidification, did notinhibit regulation of pitcher free calcium, suggested that itoccurs as a result of calcium exchange sites in the pitcherwalls. The data are discussed in relation to the physiologyof Sarracenia pitchers and to the usefulness of the pitcheras a model for free calcium in the higher plant apoplast. Sarracenia purpurea L., carnivorous plant, pitcher, free calcium, plant apoplast     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C     

李氏大足蝗线粒体全基因组序列分析

采用长距PCR 扩增及保守引物步移法测定并注释了李氏大足蝗( Aeropus licenti Chang)的线粒体基因组全序列。结果表明,李氏大足蝗的线粒体基因组全长15 597 bp,A+T 含量为74.8%,37个基因位置与飞蝗的一致,基因间隔序列共计17处105 bp,间隔长度从1～21 bp不等;有10对基因间存在58 bp重叠,重叠碱基数在1～17 bp之间。13个蛋白质编码基因中找到4种可能的起始密码子;有12个基因在基因3'端找到完全的TAA或TAG 终止密码子,只有ND5基因终止密码子为不完整的T。除tRNA^Ser（AGN）外,其余21个tRNA基因的二级结构均属典型的三叶草结构。tRNA^Ser（AGN）的DHU臂缺失,在相应的位置上只形成一个环。预测的lrRNA二级结构总共有6个结构域（结构域Ⅲ缺失）,47个茎环结构;预测的srRNA的二级结构包含3个结构域,31个茎环结构。A+T 丰富区长度为712 bp。     

A Rapid Method for the Determination of the Leaf Area of Oxalis corniculata L. and Tephrosia purpurea (Linn.) pers.

A rapid simple method has been described for the determinationof leaf area of Oxalis corniculata L. (Oxalidaceae) and Tephrosiapurpurea (Linn.) pers. (Leguminoseae) by evaluating area = a²x0.866and a²x0.335 respectively for the two plants, where a representsa single linear measurement. The relation has been derived afterintegrating the equation of the curve r² = a² cos 2 coveringthe area of each leaflet. Areas of a large number of leaflets have been determined withthe help of two methods (1) Planimeter method, (2) Evaluationmethod. The significance of the difference between the two valuesfor a particular leaf has been statistically examined and itappears that the former method can be replaced by the latterin the case of these plants.     

The plankton of a large oligotrophic freshwater Antarctic lake

The planktonic community of Crooked Lake, a large freshwaterlake in the Vestfold Hills, Antarctica was investigated duringthe austral summer in 1990. Very low levels of chlorophyll aranging between mean values of 0 29 and 1.8 µl^–1were recorded. The phytoplankton was largely made up of colouredflagellates, including single species of Chlamydomonas, Ochromonasand Pendimum, which occurred in low concentrations (23.8x 10²–47.3x10² l^–1). Heterotrophic colourless flagellates, includingParaphysomonas vestita, were also relatively sparse (2.1x 10²–21.3x10²l^–1). Ciliated protozoans were particularly poorly represented.Only three species occurred reaching densities of 1001^–1,and among them the mixotrophic species Strombidium vinde wasthe most common. A single species of heliozoan Actinosphaeriumand relatively large numbers of naked amoebae were the sarcodinerepresentatives The protistan community and the bacteria wereconcentrated into microbial consortia associated with floesof paniculate organic matter probably derived from the benthicalgal mat. Of the two microcrustacean zooplankters recordedfrom the lake only Daphniopsis studeri was found breeding inthe plankton in very low numbers. The behavioural and physiologicaladaptations of the organisms inhabiting this extremely oligotrophicenvironment are discussed.     

The NADP$-specific isocitrate dehydrogenase of Rhodospirillum rubrum

The NADP^$-specific isocitrate dehydrogenase was partially purifiedfrom photosynthetically-grown Rhodospirillum rubrum. The pHoptimum is between 7.5 and 9.0 in phosphate buffer. The apparentKm is 3.1x10^–5 M for isocitrate, 5.1x10^–5 M forNADP^$, 1.7x10^–5 M for manganese, 1.5x10^–4 M formagnesium, and 3.5x10^–3 M for inorganic orthophosphate.Arsenate exerts a slight inhibition. The Q₁₀ between 17.5°Cand 40°C is 1.62, and the energy of activation at 25°Cis 9.74 Kcal/mole. Glyoxylate and oxalacetate cause concertedinhibition of the enzyme activity. Various nucleotides inhibitthe activity. The kinetics of inhibition by ATP was found tobe mixed type with respect to NADP^$ and isocitrate, the K_i valuesbeing 1.17x10^–3 M and 1.10x10^–3 M respectively.The inhibition between ATP and orthophosphate is competitivewith a K_i of 10^–4M. Thiol binding reagents are inhibitory;this inhibition is reversed by cysteine or reduced glutathione. (Received October 1, 1971; )