A simple method for identification of S phase nuclei in Vicia faba root meristems using BrdUrd labeling and indirect immunofluorescence (comparison with 3H-thymidine incorporation)

A modified immunocytochemical method for identification of S-phase cells in root meristems is desribed. The technique combines incorporation of BrdUrd, rapid isolation of cell nuclei and indirect immunocytochemical detection of BrdUrd-labeled areas of chromatin using monoclonal anti-BrdUrd (I) and FITC-conjugated (II) antibodies. The labeling indexes calculated using ³H-thymidine autoradiography and BrdUrd-immunofluorescence in root meristems of Vicia faba subsp. minor were found comparable. However, the increased sensitivity of fluorescent staining reveals an enhanced complexity of visible structure emerging during successive periods of S-phase, allowing for discriminating not only quantitative but also qualitative aspects of nuclear labeling patterns characteristic for specific periods of DNA replication.     

The effect of water depth on bacterial numbers,thymidine incorporation rates and C:N ratios in northeast Atlantic surficial sediments

The effect of water depth on bacterial biomass and their ability to synthesise DNA, by measuring their rate of [³H]-thymidine incorporation, was investigated in the northeast Atlantic at three sites of varying water depth (1100–3580 m) and sediment characteristics. Thymidine incorporation rates (y) in surficial sediments varied between 0.028 and 1.44 pmol h^–1 g^–1 and showed an exponential relationship with depth (x) according to the equation y= 2.05e^–0.0011x (r=0.9830 for n=7, P<0.001). However, this relationship failed when a layer of phytodetritus was found overlying the surface sediment and [³H]-thymidine incorporation rates increased by 80–339%. In contrast, bacterial numbers varied between 1.09 and 11.96 × 10⁸ cells g^–1 (dry weight) and showed no significant relationships with water depth or sediment POC/TN content. Significant exponential relationships were also found between water depth (x) and the POC (y ₁) and total nitrogen (TN, y ₂) content of surficial sediments according to the following equations: where y ₁ = 719e^–0.0003x (r=0.8700 for n=9, P<0.01) and y ₂ = 76e^–0.0002x(r=0.7582 for n=9 P<0.02). These relationships were irrespective of the presence or absence of an overlying layer of phytodetritus. This suggests that the POC and TN content of these surficial deep sea sediments is directly related to the flux of material through the water column, which significantly impacts bacterial production.     

Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA

Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10⁵ cells g^–1 day^–1. These rates are 2–1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed.     

Effect of garlic bulb extract on the growth and enzymatic activities of rhizosphere and rhizoplane fungi

Eighteen fungal species were isolated from rhizospheric soil and rhizoplane samples of three plant crops in southern Iraq. The fungal isolates were examined for the activities of four enzymes (amylase, cellulase, phenoloxidase, and protease), as well as their growth, against crude garlic extract added to the culture agar medium. A high reduction or inhibition of enzymatic activities was observed for the fungi treated with garlic extract compared with untreated fungal cultures. However, most of the species showed inhibition of enzymes due to the effect of garlic extract. The growth of the fungal species was also remarkably reduced by the garlic extract.This revised version was published online in October 2005 with corrections to the Cover Date.     

Root exudation and rhizoplane bacterial abundance of barley (Hordeum vulgare L.) in relation to nitrogen fertilization and root growth

The abundance of bacteria in the rhizoplane of barley varieties was investigated at different soil nitrogen levels. Increased amendments of nitrogen resulted in higher bacterial numbers in the rhizoplane of barley seedlings of different varieties. A negative correlation was found between nitrogen level in the soil and the growth rate of the seedling roots. The effect of nitrogen on the bacterial abundances could be indirect through changed root growth and thereby changed exudation. The exudation of soluble organic carbon componds from barley seedling roots were measured in hydroponic culture. The effect of natural variation in root growth rate and of different concentrations of nitrogen in the nutrient solution was investigated. The amount of exudates consituted 2–66% of the dry weight increase in root biomass, depending on the root growth. Slower growing roots released considerably more organic carbon per unit root weight than faster growing roots. The variation in root exudation appeared to be mainly explained by differences in root growth, rather than of the nitrogen concentration in the nutrient solution. A significantly higher exudation rate was found during day time compared to night.     

Effect of 8-hydroxyquinoline on the uptake of uridine and incorporation into RNA

8-hydroxyquinoline has been previously used as an inhibitor in studies on porphyrin metabolism, where it is thought to act by chelating iron. It is shown that this compound also rapidly inhibits uridine uptake of seedlings or cotyledons of the crucifer Matthiola incana R.Br. RNA synthesis is also affected but the inhibition is not as severe as reported for fission yeast.Abbreviations oligo (dT)-cellulose cellulose with oligo-deoxythymidylic acid attached - poly (A) polyadenylic acid     

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-thymidine and differential gating method

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, ³H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Mortality of marine bacterial strains in seawater

As an approach for assessing the dynamics of bacterial population in seawater, the survival of five isolated marine bacteria strains was assessed by the disappearance of radioactivity in the cold trichloroacetic acid (TCA)-insoluble fraction from a previously³H-labeled culture. Metabolic activity during survival experiments was assessed by the measurement of electron transport system (ETS) activity. Fractionated filtration was used to assess the grazing mortality. The particulate fraction that passed 2.0 m and was retained in 0.2 m was the main cause of mortality.     

Nitrogen incorporation and remobilization in different shoot components of field-grown winter oilseed rape (Brassica napus L.) as affected by rate of nitrogen application and irrigation

The seasonal course of nitrogen uptake, incorporation and remobilization in different shoot components of winter oilseed rape (Brassica napus L.) was studied under field conditions including three rates of ¹⁵N labelled nitrogen application (0, 100 or 200 kg N ha^-1) and two irrigation treatments (rainfed or watered at a deficit of 20 mm). The total amount of irrigation water applied was 260 mm, split over 13 occasions in a 7-week-period ranging from 1 week before onset of flowering until 4 weeks after flowering.Nitrogen application and irrigation increased plant growth and nitrogen accumulation. Irrespective of N and irrigation treatment more than 50% of total shoot N was present in the stem when flowering started. At the end of flowering, pod walls were the main N store containing about 30–40% of shoot N. The quantities of N remobilized from stems and pod walls amounted in all treatments to about 70% of the N present in these organs at mid-flowering. At harvest, stem and pod walls each contained about 10% of total shoot N, the remaining 80% being incorporated into seeds. The main component contributing to the response of seed N accumulation to nitrogen application and irrigation was pods in axillary racemes. Up to 20 kg N ha^-1, corresponding to about 10% of final shoot N content, was lost from the plants by leaf drop.Irrigation increased the recovery at harvest of applied N from 30% to about 50%, while the level of N application did not affect the N recovery. ¹⁵N labelled (fertilizer derived) nitrogen constituted a greater proportion of the N content in old leaves than in young leaves and increased with age in the former, but not in the latter. Relative to soil N, fertilizer derived N also contributed more to the N content of vegetative than to that of reproductive shoot components. Small net changes in shoot N content after flowering reflected a balance between N import and export, leading to continuous dilution of ¹⁵N labelled N with unlabelled N.     

Discrimination among cultivars of rapeseed (Brassica napus L.) using DNA polymorphisms amplified from arbitrary primers

RAPDs (Randomly Amplified Polymorphic DNAs) were used to discriminate among 23 cultivars of oilseed rape (Brassica napus) selected from several breeding programs. A set of 100 random sequence 10-mer primers were tested, of which 70 produced bands and 22 showed evidence of polymorphism. A selection of six primers produced 23 polymorphic bands of between 300 to 2200 base pairs in size, sufficient to distinguish between the cultivars. An analysis of seed of five cultivars obtained from four different sites showed stability of banding pattern over source of seed. The analysis was repeated using four different thermocyclers, each of which produced the same band pattern. UPGMA cluster analysis indicates that the relationships among some of the cultivars is closer for those from the same breeding program than for those from different programs. The results of this study show that RAPDs can be used as a method of identification for oilseed rape cultivars.Contribution number 941     

DNA sequences of the two homoeologous SLR1 genes of Brassica napus cv Westar

The DNA sequence data reported have been lodged in the Genbank, EMBL and DDBJ databases under the accession numbers Z21609 and Z26914     

Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hupc mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme

A double mutant (JH103K10) was created from hydrogenase constitutive mutant (JH103) by replacement of a chromosomal 0.60 kb nickel metabolism related locus with a kanamycin resistance gene. The double mutant required 10 to 20 times more nickel (Ni) to achieve near parental strain levels of hydrogenase activity. In the absence of nickel, both JH103K10 and JH103 synthesized high levels of (inactive) hydrogenase apoprotein (large subunit, 65 kDa). With nickel, the double mutant JH103K10 synthesized the same level of hydrogenase apoenzyme (65-kDa subunit) as the JH103 parent strain; however, whole cell hydrogenase activity in JH103K10 was less than half of that in JH103, and the CPM (due to ⁶³Ni in hydrogenase) of membranes and the calculated ratio of nickel per unit of hydrogenase enzyme of the double mutant were 40% of that in JH103. Therefore, the difference in hydrogenase activities between the double mutant and the Hup^ck strain can be accounted for by different abilities of the strains to incorporate nickel into the hydrogenase apoenzyme. The addition of nickel ions to previously Ni-starved and then chloramphenicol-treated Bradyrhizobium japonicum whole cells (JH103 and JH103K10) resulted in (an in vivo) restoration of hydrogenase activity, suggesting that the apoprotein synthesized in the Ni-free cultures could be activated by addition of nickel even in the absence of protein synthesis. The extent of reconstitution of active hydrogenase by nickel was greater in the absence of chloramphenicol. Hydrogenase apoprotein could not be activated by nickel in vitro even with the addition of ATP. The successful in vivo but not in vitro results suggest that enzymatic but cell-disruption labile factors are required for Ni incorporation into hydrogenase.     

Immunolocalization and histochemical evidence for the association of two different<Emphasis Type="Italic"> Arabidopsis</Emphasis> annexins with secretion during early seedling growth and development

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following ³H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.     

Regional differences in renewal rates of fibroblasts in young adult female mice

Summary Young adult female mice were given a total of 90 intraperitoneal injections each of (methyl-³H)thymidine (³HTdR) at intervals of 8 h over 30 days to establish renewal rates of fibroblasts in various locations. Radioautographs prepared from punch biopsy material of the ears, taken repeatedly during the labeling procedure, revealed an approximately linear increase of labeling indices of dermal fibroblasts with time. Labeling indices of fibroblasts at the end of repetitive injections of ³HTdR differed depending on the site and/or type of connective tissues examined. Low values were obtained for fibroblasts in the leptomeninx (3.9%), the tracheal wall (4.4%), the achilles tendon (5.8%) and the dermis of the ear (6.5%), while higher values were registered for fibroblasts located in the lower half of the abdominal dermis (12.3%), peritendinous sheaths (12.6%), the interstitial connective tissue of the thigh muscles (12.9%), the submucosa of the colon (23%), the fibrous capsule of the adrenals (25.7%), and the upper half of the abdominal dermis (26%). These regional differences, with the exception of the skin and possibly of the tracheal wall, did not correlate with local temperature. Possible additional factors influencing fibroblast renewal rates may include the type of connective tissue, the degree of vascularization, mechanical stress and hormonal action. Estimated turnover times, based on the assumptions of a DNA synthesis time of 6 h or more and a linear increase of labeling indices as a function of time of repetitive labeling, range from about 90 to more than 700 days. The higher values approximate the median life span of the mouse strain used.This work was supported by the Swiss National Science Foundation     

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example

Summary The use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F₂ population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.     

Towards the genetic engineering of triacylglycerols of defined fatty acid composition: major changes in erucic acid content at the sn-2 position affected by the introduction of a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii into oil seed rape

A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.