A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of Citrus explants throughout the annual cycle and its regulation by ethylene

Leaf senescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) degradation in orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] explants have been investigated. Explants consisted of a segment of stem (ca 15 cm) and 5 mature leaves. In vitro RuBP carboxylase degradation was determined by culturing the explants in water for different periods of time (3 days usually) and quantifying the two RuBP carboxylase subunits in the extracts following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro RuBP carboxylase degradation was estimated by autodigestion of leaf extracts and SDS-PAGE. The extent of in vivo RuBP carboxylase degradation in explants cultured under 16 h light/8 h dark photoperiod varied throughout the year and showed a cyclic behaviour correlated with the growth cycle of Citrus. The highest proteolytic activity both in vivo and in vitro was found in explants made from April to August coinciding with the maximum vegetative growth period of the tree.
Leaf senescence and abscission could be retarded significantly at any time of the year by maintaining the explants continuously in the dark. Treatment of the explants in the dark with a continuous flow of ethylene enhanced both leaf abscission and rate of RuBP carboxylase degradation, proportionally to ethylene concentration (0.1-0.6 ppm). Ethylene-induced senescence of Citrus leaf explants in the dark appears to be a convenient model system to study the regulation of the proteolytic degradation of RuBP carboxylase.     

Tree-ring analysis and conifer growth responses to increased atmospheric CO2 levels

Summary Tree-ring data of naturally grown connifers were analyzed to evaluate the possibility of enhanced tree growth due to increased atmospheric CO₂. Tree cores were obtained from 34 sites in four different climatic regions in the northern hemisphere. In each of the four regions, the sampling sites were located along ecological gradients between the subalpine treeline and low elevations and, sometimes, the arid forest border. Growth trends after 1950, when the atmospheric CO₂ concentration increased by more than 30 l·l^-1 indicate an increase in ring-widths at eight of the 34 sites. These chronologies were from sites which moderate temperature or water stress. In four cases the growth increase in the post-1950 period coincided with favorable climatic conditions. In the remaining four cases, the growth increase exceeded the upper bound response expected from CO₂ enrichment experiments with seedling conifer species. Therefore, increased growth in any of the tree-ring chronologies examined could not be solely attributed to higher atmospheric CO₂ concentrations.Major financial supporters: Swiss National Science Foundation (application no. 1.869-0.83); Swiss Federal Institute of Forestry Research, 8903 Birmensdorf, Switzerland; other financial supporters: Carbon Dioxide Research Division, U.S. Department of Energy under subcontract no. 11X-57507V with Martin Marietta Energy Systems, IncOperated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-840R21400 with U.S. Department of Energy     

Voltage dependence of Na/K pump current inXenopus oocytes

Summary Stage V and VI (Dumont, J.N., 1972.J. Morphol. 136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 m dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (–40 mV). The resulting membraneI–V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI–V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI–V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI–V relationship. DHO differenceI–V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 A cm^–2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI–V relationships do not show a region of negative slope over the voltage range –110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Effect of exogenous hormones on the ovulation of primary and secondary oocytes in LT/Sv strain mice

LT/Sv strain mice ovulate both primary and secondary oocytes. These are fertilizable and give rise to digynic triploid and normal diploid conceptuses, respectively. A previous study [Kaufman and Speirs, 1987] had indicated that just over 20% of embryos recovered on the 10th day of gestation from spontaneously ovulating females had a triploid chromosome constitution. This value was considerably lower than might have been expected by extrapolation from earlier studies in which LT/Sv mice had been given exogenous gonadotrophins. In the present study, therefore, cytogenetic analysis of fertilized eggs was performed at the first cleavage mitosis in (1) spontaneously ovulating females mated to F1 hybrid males, and (2) superovulated females mated to similar males. Additional females from group (1) were autopsied on the 10th day of gestation, and the ploidy of embryos isolated at this stage of gestation was determined. Exposure to exogenous gonadotrophins significantly increased the proportion of eggs that were ovulated as primary oocytes (34.4%), compared to the situation observed following spontaneous ovulation (24.4%). All the triploids encountered in both series were of the digynic type and characteristically (for LT/Sv mice) had an oocyte-derived set with 40 chromosomes present, and a sperm-derived set containing 20 chromosomes. Similar numbers of eggs were recovered from spontaneously ovulating females on the 1st and 10th days of gestation, and the incidence of triploidy observed on the 10th day was 22.1%. The influence of exogenous hormones in increasing the “spontaneous” level of triploidy in LT/Sv and in other strains of mice is briefly reviewed.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.