Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

The genetic control of seven isozymic loci in Vicia faba L. Identification of lines and estimates of outcrossing rates between plants pollinated by bumble bees

A simplified and non-destructive method using starch gel electrophoresis has been developed on seeds to identify inbred lines of Vicia faba and assess outcrossing rates and gene dispersal in pollination experiments. Six enzyme systems (Alcohol dehydrogenase, Aspartate aminotransferase, Glucose-6-phosphate isomerase, Isocitrate dehydrogenase, Phosphogluconate dehydrogenase and Shikimate dehydrogenase) were analysed from parental lines, crosses performed between lines bearing dissimilar isozyme patterns in pollination cages with Bombus and F₂ progenies obtained from manual selfing of F₁ hybrids. The allozymes at each of the seven studied loci segregated in the expected Mendelian fashion and behaved in a co-dominant manner except for the Adh-2 locus where the only variant was a null allele. No evidence of genetic linkage was observed between at least 13 of the 15 pairs of the studied loci. Percentage of cross fertilisation by Bombus between seven pairs of inbred lines ranged between 1.7% and 28.3%. Pollen transfer between a donor line and a recipient line by two species of Bombus did not lead to differences in outcrossing rates (both about 8%). The new PGD marker with two loci at three alleles each is particularly discriminating and valuable in pollination studies and breeding of V. faba.     

Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA₁₂ yielded GA₁₃, GA₄₃ and 12α-hydroxyGA₄₃ as major products, GA₄, GA₃₇, GA₃₉, GA₄₆ and four unidentified compounds as minor products. The intermediates GA₁₅, GA₂₄ and GA₂₅ accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA₄₃ was inferred from its mass spectrum and by its formation from both GA₃₉ and GA₄₃. Gibberellin A₃₉ and 12α-hydroxyGA₄₃ were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A₁₂-aldehyde was metabolised to essentially the same products as GA₁₂ but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A₅₃, formed from GA₁₂ by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA₁ GA₂₃, GA₂₈, GA₄₄, and putative 2β-hydroxyGA₂₈. Minor products were GA₁₉, GA₂₀, GA₃₈ and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA₅₃) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553–558), GA₁, GA₈, GA₂₅, GA₂₈, GA₃₆, GA₄₈ and 12α-hydroxyGA₄₃ were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA₄₃ and GA₁ (or GA₈), respectively, as their end products. Evidence for endogenous GA₂₀ and GA₂₄ was also obtained but it was less conclusive due to interference.     

石菖蒲中一新倍半萜

石菖蒲（Acorus gramineus Soland）的精油经GC/MS/DS检测,硅胶加压柱层分离,得到一新的倍半萜,其在精油中的含量达60.30％,通过波谱方法确定了结构,并命名为石菖蒲酮（gramenone）。     

半枝莲中二萜内酯和黄酮化合物的分离和鉴定

半枝莲（Scutellaria barbata D.Don）为唇形科黄芩属植物,全草入药,具有清热解毒,化瘀利尿,消肿止痛和抗癌等功效。国内学者报道从全草中分得红花素（carthamidin）、异红花素（isocarthamidin）、印黄芩甙（scutellarein）、β-谷甾醇（β-sitosterol）、硬脂酸（stearic acid）和生物碱。台湾学者从中分离得汉黄芩素（wogonin）、5-羟基-7,8-二甲氧基黄酮（5-hydroxy-7,8-dimethoxyflavone）、半枝莲素（Rivularin）。我们从全草的乙醇提取液中分得两个化合物,经鉴定为汉黄芩素、新穿心莲内酯,该内酯在本植物中属首次发现。