Recombinant Components of the EcoRII Restriction–Modification System: Restriction Endonuclease Can Interact with DNA · RNA Duplexes

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction–modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multicopy plasmid. Recombinant R·EcoRII bound with hybrid DNA·RNA duplexes.     

The temperature-dependent duration of development and parasitism of three cereal aphid parasitoids, Aphidius ervi, A. rhopalosiphi, and Praon volucre

Temperature dependencies were established for the egg-to-mummy and mummy-to-adult phases, for mummy mortality, and for parasitism of Aphidius ervi Haliday, Aphidius rhopalosiphi De Stefani-Perez, and Praon volucre (Haliday) (Hymenoptera, Aphidiidae), three parasitoids of Sitobion avenae (Fabricius) (Homoptera, Aphididae), at 8°C, 12°C, 16°C, 20°C, and 25°C on winter wheat (cv. Haven). A physiological model described temperature-dependent development over the full temperature range, whereas a linear model was fitted for data above 8°C and used to estimate the lower temperature thresholds and day-degrees (° D) required for development. The thresholds for A. ervi were 2.2°C for egg-mummy development and 6.6°C for mummy-adult development, those for A. rhopalosiphi were 4.5°C and 7.2°C, and those for P. volucre were 3.8°C and 5.5°C. The time to develop into mummies and adults differed significantly between the three species: A. ervi development into mummies required an average of 159 ° D, while development into adults took an average of 73 ° D. The corresponding average times required for A. rhopalosiphi and P. volucre to develop mummies were 124° D and 126° D, while their development into adults required an average of 70° D and 150° D, respectively. Mummy mortality was 25–35% at 8°C and less at the higher temperatures tested, but began to increase again at 25°C, showing a quadratic relationship between mortality and temperature. Parasitization was very low or, in the case of P. volucre, absent up to 12°C and thereafter increased with increasing temperature. The relationship between parasitization, recorded as percent aphids mummified, and temperature was linear at the temperatures tested and depended on species. A. ervisuperparasitized 11.1% aphids at 20°C and 16.6% aphids at 25°C, whereas superparasitism was low in A. rhopalosiphi and absent in P. volucre. From 16°C to 25°C the P. volucre sex ratio increased. For A. ervi and A. rhopalosiphi there was no trend with temperature, but at 20°C and 25°C it was close to even. Field data for 1996 and 1997 allowed for a comparison of actual and expected emergence of overwintering mummies. In both years, parasitoids were predicted to have emerged from overwintering mummies well in advance of the onset of aphid infestation, and more than a month earlier than the first parasitized aphids were found in winter wheat. Observations from trap plants in other crops supported the predictions of the models. Other factors that can affect biological control by cereal aphid parasitoids are discussed.     

Porosity and available water of temporarily waterlogged soils in a Quercus robur (L.) declining stand

Pedunculate oak (Quercus robur L.) is particularly sensitive to decline in clayey soils presenting a high-perched temporary water table. These soils induce two successive constraints in one-year cycle: water excess (and hypoxy) in winter and early spring, and water shortage in summer (water stress being more restrictive to oak). We determined the porosity and water properties of temporarily waterlogged clayey soils supporting forest stands of decliningQuercus robur trees in a 101yr-old oak stand in Belgium (50°06N, 4°16E). Roots unevenly colonized the soil down to 1.6m: fine roots (diameter<5mm) were mostly distributed on the surface horizons (0–0.3 m) and around 1.3m deep, despite dense clayey horizons appearing at 0.35m depth. Clay content below this depth reached 46–51. Illite and vermiculite were the dominant clay minerals. The clayey horizons exhibited marked shrink–swell properties: bulk density at 30kPa increased from 1.41 to 1.88gcm^–3 from the surface to 2m depth. There was also evidence of hypoxic conditions caused by water saturation of pore space in the rooting zone from October to mid-April. Extractable water (EW), calculated between moisture tensions of 5 and 1600kPa was 152.8mm. The level of perched temporary water table strongly depended on the seasonal rhythm of water uptake by trees and on the shrink–swell behaviour of soil.     

Importance of topography and soil texture in the spatial distribution of two sympatric dipterocarp trees in a Bornean rainforest

Relationships between spatial distributions and site conditions, namely topography and soil texture, were analyzed for two congeneric emergent trees, Dryobalanops aromatica and Dryobalanops lanceolata (Dipterocarpaceae), in a tropical rainforest in Sarawak, East Malaysia. A 52-ha permanent plot was divided into 1300 quadrats measuring 20m×20m; for each Dryobalanops species, the number and total basal area of trees 1cm in d.b.h. were compared among groups of quadrats with different site conditions. Because spatial distributions of both Dryobalanops and site-condition variables were aggregated, Monte-Carlo permutation tests were applied to analyze the relationships. Both single and multifactor statistical tests showed that the density and basal area distributions of the two species were significantly non-random in relation to soil texture and topographic variables. D.aromatica was significantly more abundant at higher elevations, in sandy soils, and on convex and steep slopes. In contrast, D.lanceolata preferred lower elevations and less sandy soils. In the study plot, there were very few sites (3 of 1150 quadrats tested) where the models of Hayashis method predicted the co-occurrence of the two species. These results suggest that between-species differences in habitat preferences are so large that they alone explain the spatially segregated distributions of these two species within the 52-ha study plot.     

MHC restriction of male-antigen-specific T helper cells collaborating in antibody responses

By priming female C57BL/6 mice with syngeneic male spleen cells and enriching inguinal and paraaortic lymph node cells in long-term culture (LTC) by repeated restimulations, H-Y-specific T helper cells can be produced. In response to male spleen cells carrying I-Ab antigens these cells activate antigenexpressing B cells to secrete polyclonal antibody. Before the end of the second week in LTC it was impossible to detect any helper activity. Induction of plaque-forming cells (PFC) also requires simultaneous recognition of antigen and I-A-encoded determinants in the stimulator-responder spleen-cell population. The testing of spleen cells fromH-2 recombinant strains as stimulator-responders to anti-H-Y helper T cells of C57BL/6 origin also revealed that other genes, telomeric toI-A, control the magnitude of both specific T-cell proliferation and helper-dependent B-cell activation.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Larval development and food habits of the marbled parrotfish, <Emphasis Type="Italic">Leptoscarus vaigiensis</Emphasis>, associated with drifting algae

The larval development and food habits of the marbled parrotfish, Leptoscarus vaigiensis (Scaridae) associated with drifting algae were studied. In this study, 628 L. vaigiensis of various developmental stages ranging from postflexion larvae (9.4mm in standard length, SL) to adults (192.0mmSL) were sampled from drifting algae at two fishing ports in Nakagusuku Bay of Okinawa Island. In 3969 fish comprising 65 taxa in 34 families of Teleostei collected with drifting algae, L. vaigiensis occupied 15.8% of samples and occurred generally throughout the whole year. A large number of L. vaigiensis were collected from July to October accompanied by an occurrence of drifting algae composed of Sargassum spp. Larvae and early juveniles ranging from 11.1 to 14.9mmSL appeared sporadically throughout the year, and postflexion larvae 11mmSL occurred from July to November. Their food shifted from planktonic copepods in postflexion larvae and juveniles ranging from 10.0 to 14.9mmSL to seaweed in the juveniles ranging from 15.0 to 24.9mmSL. Furthermore, adults and young over 25mmSL fed almost exclusively on seaweed, with Sargassum spp. constituting the drifting algae. These facts indicate that drifting algae may have a role concerning food and habitat, and may act as a nursery for L. vaigiensis.     

Rapid micropropagation of <Emphasis Type="Italic">Curcuma longa</Emphasis> using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Multiple shoots of Curcuma longa were induced by culture of bud explants for 1week in Murashige and Skoog (MS) liquid medium supplemented with 72.64M thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8weeks. The regeneration rate was up to 11.4±1.7shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

Root traits, nutrient uptake, multi-location grain yield and benefit–cost ratio of two lentil (Lens culinaris, Medikus.) varieties

Lentil is a protein-rich pulse, grown mainly in developing countries as a rain-fed crop in nutrient-poor soils. Hence, the importance of root traits for efficient capture of soil nutrients and water can be crucial to its economical yield. Little is known about the lentil root system and even less about its relationship to grain yield. We compared the root system of two Bangladeshi lentil varieties, Barimasur-3 (BM-3) and Barimasur-4 (BM-4), in a pot experiment and related it to their multi-location grain yield in the fields. BM-4 maintained faster root development both at an early growth stage (20days after sowing) and at flowering (60days) compared to BM-3. The roots of BM-4 penetrated the 25cm depth of the soil profile after 19±1days and while those of BM-3 took 24±2days to reach the same depth. The roots of BM-4 were covered with denser (26±3mm^–1) and longer (0.48±0.11) root hairs than BM-3 (density 17±2mm^–1, length 0.32±0.09mm). The differential presence of root hairs increased the effective length of root system of BM-4 by 12 times and that of BM-3 by five times. The lentil varieties did not differ in their ability to induce pH change and acid phosphatase activity in rhizosphere. In the pot experiment, the uptake of macro-nutrients (K, P, Ca, and Mg) as well as micro-nutrients (Fe, Mn, Zn, Cu, B and Mo) by BM-4 was significantly higher, compared to BM-3. The varieties produced the same amount of shoot biomass. At five of six agro-ecological distinct field locations in Bangladesh, BM-4 gave significantly higher (10–20%) grain yield than BM-3. Linked with the higher grain yield, the benefit-cost ratio (BCR) of BM-4 was 3.14 and that of BM-3 were 2.62, indicating that BM-4 provided better return per unit investment, compared to BM-3, supported by the better root morphology and higher nutrient uptake. This may be one of the reasons supporting the popularity and preferred adoption of BM-4 among the Bangladeshi farmers, who grow lentil mainly on nutrient-poor soils. The results indicate the benefits of selection and breeding for superior root traits for better agro-economics.     

Composition and mean residence time of molecular weight fractions of organic matter extracted from two soils under different forest species

The organic matter extracted from various mineral horizons of two forest soils, one under silver fir (Abies alba Mill.), the other under European beech (Fagus sylvatica L.), was fractionated by dialysis into three fractions, 100–1000, 1000–8000, and >8000Da. On a C basis, in all horizons the recovered organic matter amounted to less than a half of the total and was mainly composed of molecules >8000Da. The 100–1000Da fraction had a principal elemental composition profoundly different from the other two fractions, which, instead differed from each other significantly only for the S content and the molar ratio of C with N. No significant difference in this regard was found between soils. The richness in O and some typical absorption bands in the FT-IR spectra indicated that the 100–1000Da fraction had a lot of carboxyl moieties. The spectroscopic (¹³C NMR) investigation showed that the 1000–8000 and >8000Da fractions had a prevalently aliphatic nature and signals attributable to polysaccharides (O-alkyl C) revealed overall a high presence of non-humic biopolymers. These latter were significantly more abundant, suggesting a lower degree of humification, in the >8000Da fraction than in the 1000–8000Da fraction. Comparing soils, that under beech appeared significantly richer in O-alkyl C than that under fir. The organics extracted from the A horizon of both soils had positive ¹⁴C values, indicating recent synthesis mainly due to the present forest cover. The mean residence time (MRT) of the combined 100–1000Da and 1000–8000Da fractions and the >8000Da fraction increased with depth, even to about 5000 years in the more than 1-m deep BC horizons under beech. In some cases, and especially in the soil under fir, despite higher values of ¹³C denoting stronger microbial decomposition, the 100–8000Da fraction showed a higher MRT than that of the >8000Da fraction, perhaps due to its ascertained lower content of non-humic biopolymers.     

Improvement of growth and camptothecin yield by altering nitrogen source supply in cell suspension cultures of Camptotheca acuminata

Nitrate at 70m gave the highest biomass of Camptotheca acuminata in suspension culture in MS medium, but a NH₄ ⁺/NO₃ ^– molar ratio of 5:1 (giving a total of 40 m N) gave the maximum camptothecin yield. A two-stage flask culture system was established to improve culture efficiency; cell dry weight, camptothecin content and yield was increased by 30%, 280% and 340%, respectively when compared with those of control, reaching up to 36g l^–1, 0.36mgg^–1, and 12.8mgl^–1, respectively.     

Production of xylanases by immobilized Aspergillus sp. using lignocellulosic waste

In shake flasks immobilized Aspergillus terreus and Aspergillus niger produced 29IU/ml, 26.7IU/ml xylanases at 10mg/ml, 14mg/ml wheat bran concentration after 48 and 60h of incubation at 37°C respectively. In repeated batch fermentation of immobilized Aspergillus sp. the same biocatalyst could be used for three successive cycles.     

Ecological classification of Nigerian mangroves using soil nutrient gradient analysis

Direct gradient analysis was used to relate the structure of mangrove communities to soil nutrient gradients. The predominant cations in the alluvial soils were magnesium and calcium, the values ranging from 8.6±0.9 to 24.6±2.0me per 100g. Organic carbon was high in the soils, ranging from 3.5% to 10.4%. All soil nutrients varied seasonally, in response to wet and dry periods of the climate. Correlation of species with soil nutrients indicated the nutrient status of the soils to be best defined in terms of calcium for the A stratum (species > 3m tall), calcium/postassium for the B stratum (species 1–3m tall) and magnesium for the C stratum (species < 1m tall). Ecological group classification shows that Avicennia africanaand Nypa fruticansoccurred almost exclusively at the highest calcium values of 20.8 and 17.6me per 100g, respectively. Acrostichum aureumand Sesuvium portulacastrumare insensitive to the magnesium gradient. The analysis revealed an overlapping range of occurrences for most of the species although with varying ecological optima along the gradient.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Components of spatial patterning in a serpentine grassland

We analyzed the components of spatial patterning in species abundance in a diverse grassland dominated by early season, annual forbs. Species abundance, soil depth and gopher disturbances were measured by means of a nested spatial design on two 8m×20m plots that differed in the amount of larger scale variability in soil depth. Species distributions at the scale of the 10cm×10cm quadrat were highly clumped, with a decline in clumping with mean abundance. Even with the effect of abundance on clumping removed, species showed differences in the degree of clumping and these differences were consistent between the two plots. The abundance of the species and the frequency of disturbance correlated weakly to moderately with soil depth. Semivariance analyses indicated that the most common species all showed complex spatial patterning at a range of scales. Some of this variation corresponded to patterns of soil depth variation and patterns of gopher disturbance; however, a large amount of the spatial patterning in species abundance remains unexplained.     

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis

Summary. Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3±4.1µm) than those developing 2 PPBs (49.8±4.1µm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42µm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.Correspondence and reprints: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Soil characteristics and nitrogen resorption in Stipa krylovii native to northern China

Nitrogen (N) resorption from senescing tissues enables plants to conserve and reuse this important nutrient. As such, it is expected that plant species adapted to infertile soils could have a higher N-resorption efficiency (percentage reduction of nitrogen between green and senescing tissues) and/or higher N-resorption proficiency (absolute reduction of nitrogen in senescing tissues) than those adapted to fertile soils. To test this hypothesis, we investigated the relationships among soil characteristics (total N, nitrate-N, ammonium-N, pH and moisture) and N resorption in Stipa kryloviiRoshev., a species occurred widely in natural grasslands of northern China. N contents in green and senescing tissues were 6.7±0.1 and 3.3±0.1 mg g^–1, respectively. The mean value of N-resorption efficiency was found to be 72.1%. The N-resorption efficiency in S. kryloviiwas independent of soil characteristics. The N-resorption proficiency in S. kryloviiwas dependent on soil nitrate- and ammonium-N, but it was relatively independent of soil total N. The N-resorption proficiency was negatively correlated with soil pH and moisture. There was a positive correlation between N concentration in green tissues and resorption efficiency. However, N-resorption efficiency was not correlated significantly with N concentration in senescing tissues. These results indicate that the intraspecific variation in N resorption of Stipa kryloviiRoshev. is associated with soil regimes and that higher N resorption on N-poor soils is an adaptive strategy for S. kryloviito maximize N use under conditions of limited N supply.     

Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology

Theaflavins were synthesized from tea polyphenols extracted from green tea using an immobilized polyphenol oxidase system. To optimize the production of theaflavins, response surface methodology was applied to determine the effects of five critical variables and their mutual interactions on theaflavin biosynthesis at five levels. A total of 52 individual experiments were performed and a statistical model predicted that the highest theaflavin concentration was 0.766mgml^–1 at optimized conditions. Using these optimal parameters under experimental conditions in three independent replicates, the average value of the biosynthesized theaflavin concentration reached 0.75±0.017mgml^–1 and matched the value predicted by the modelRevisions requested 03 November 2004; Revisions received 7 December 2004